Synthetic peptides that bind to the hepatitis B virus core and e antigens

ABSTRACT

The present invention relates generally to the field of virology. More particularly, the invention relates to the discovery that peptides, which bind to the Hepatitis B virus (HBV) core and e antigens, can be used to inhibit HBV infection. Embodiments concern “binding partners”, which include peptides, peptidomimetics, and chemicals that resemble these molecules that interact with HBV core and e antigens, biological complexes having HBV core and e antigens joined to said binding partners, methods of identifying such binding partners, pharmaceuticals having binding partners, and methods of treatments and prevention of HBV infection.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part and claims priority toU.S. application Ser. No. 09/556,605, filed Apr. 21, 2000, which ishereby expressly incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of virology.More particularly, the invention relates to the discovery that peptidesthat bind to the hepatitis B virus (HBV) core and e antigens can be usedto inhibit HBV infection.

BACKGROUND OF THE INVENTION

[0003] Of the many viral causes of human hepatitis, few are of greaterglobal importance than hepatitis B virus (HBV). Approximately 300million people worldwide are chronically infected and some of thesechronically infected individuals develop severe pathologic consequencesincluding chronic hepatic insufficiency, cirrhosis, and hepatocellularcarcinoma (HCC). (See Fields Virology, third ed., edited by Fields etal., Lipponcott-Raven Publishers, Philidelphia 1996 pp. 2703 and Lee etal., Cancer, 72:2564-7 (1993)). Primary infection may be asymptomatic(e.g., in chronically infected individuals) or may result in varyingdegrees of acute liver injury. (Milich et al., Springer Seminars inImmunopathology, 17:149-66 (1995)).

[0004] HBV is unusual among animal viruses in that infected cellsproduce multiple types of virus-related particles. (See Fields Virology,third ed., edited by Fields et al., Lipponcott-Raven Publishers,Philidelphia 1996 pp. 2704). Electron microscopy of partially purifiedpreparations of HBV shows three types of particles, a 42-47 nminfectious particle (referred to as “Dane particles”), non-infectious 20nm spheres, and non-infectious 20 nm diameter filaments of variablelength. Id. at 2705-2705. The HBV genome encodes at least fivestructural proteins: the envelope or surface proteins preS1, preS2, andS (HBsAg); the polymerase; and the core or capsid antigen (HBcAg). Allthree forms of HBV particles have HBsAg, which serves as an epitope forneutralizing antibodies and is the basis for state of the art HBVdiagnostics. In contrast, only the Dane particles have HBcAg, a 21 kDphosphoprotein that is believed to be phosphorylated in vivo. Id. at2705. The HBV genome also encodes the non-structural proteins HBeAg andX. The HBcAg and the HBeAg are translated from two different mRNAs thatare transcribed from the same open reading frame. The longer of the twomRNAs encodes HBeAg. HBcAg and the HBeAg share an amino acid sequence ofapproximately 150 residues.

[0005] HBcAg is highly immunogenic in humans and mice. Investigatorshave observed that HBcAg induces B-cells to produce IgM and, thus, iscurrently classified as a partially T cell independent antigen. (Milichand McLachlan, Science, 234:1398-401 (1986)). HBcAg can also crosslinkB-cell surface receptors and membrane bound IgM on naive B-cells and, inturn, HBcAg can be taken up, processed, and presented to HBcAg-specificCD4+T cells. (Milich et al., Proc Natl Acad Sci USA, 94:14648-14653(1997)). Quite surprisingly, B-cells that are able to bind and presentHBcAg exist in great numbers in naive non-immunized mice. Theidentification of molecules that inhibit HBV infection by interactingwith HBcAg and/or HBeAg remains a largely unrealized goal.

BRIEF SUMMARY OF THE INVENTION

[0006] The invention described herein concerns the identification andmanufacture of molecules that interact with HBcAg and/or HBeAg andthereby inhibit HBV infection or modulate a host immune system responseor both. Molecules that interact with HBcAg and/or HBeAg, also referredto as “binding partners”, are designed from fragments of antibodies andother proteins that interact with HBcAg and/or HBeAg. Accordingly, anamino acid sequence corresponding to the binding domains of monoclonalor polyclonal antibodies or proteins that bind HBcAg and/or HBeAg isused as a template for the design of synthetic molecules, including butnot limited to, peptides, derivative or modified peptides,peptidomimetics, and chemicals. A preferred binding partner, forexample, is a molecule called a “specificity exchanger”, which comprisesa first domain that interacts with HBcAg and/or HBeAg and a seconddomain that has an epitope for a high titer antibody, preferably anepitope on a pathogen or a toxin. The binding partners described hereincan be manufactured by conventional techniques in peptide chemistryand/or organic chemistry.

[0007] Methods to characterize binding partners are also embodiments.The term “characterization assay” is used to refer to an experiment orevaluation of the ability of a candidate binding partner and/or bindingpartner to interact with HBcAg and/or HBeAg, inhibit HBV infection, ormodulate a host immune response. Some characterization assays, forexample, evaluate the ability of a binding partner to bind to amultimeric agent having HBcAg and/or HBeAg disposed thereon or viceversa. Other characterization assays access the ability of a bindingpartner to fix complement and/or bind to a high titer antibody.Additional characterization assays determine whether a binding partnercan effect viral infection in cultured cell lines or infected animals.Still further, some embodiments evaluate the ability of a bindingpartner to modulate a host immune system response, as measured bycytokine production and/or T cell proliferation.

[0008] Binding partners can be used as immunochemicals for the detectionof HBcAg and/or HBeAg and can be incorporated into diagnostic methodsand kits. Binding partners, preferably specificity exchangers, can alsobe incorporated into pharmaceuticals and used to treat or prevent HBVinfection. A preferred embodiment concerns a method of treating orpreventing HBV infection by identifying a subject in need andadministering said subject a therapeutically effective amount of bindingpartner.

[0009] As described herein, embodiments include a peptide that bindsHBcAg or HBeAg having about 3-50 amino acids residues. Preferably, thesequence of said peptide is selected from the group consisting of SEQ.ID. Nos. 4-45, 53, 54, 66-69, 71, and 74. Other embodiments include apeptide comprising the sequence of at least one of SEQ. ID. Nos. 1-3, apeptide comprising the sequence of SEQ. ID. No. 45, a peptide comprisingthe sequence of SEQ. ID. No. 54, a peptide comprising the sequence ofSEQ. ID. No. 74, and a peptide having a specificity domain, which bindsHBcAg or HBeAg and an antigenic domain joined to the specificity domain,wherein said antigenic domain binds a high titer antibody, preferably anepitope for a pathogen or toxin.

[0010] Related embodiments concern a peptidomimetic that corresponds toa peptide selected from the group consisting of SEQ. ID. No. 1, 2, 3,45, 54, and 74 and an isolated or purified peptide that is less than 50amino acids in length having the formula: X¹ _(n)CKASX²n, wherein “X¹”and “X²” are any amino acid and “n” is any integer, and wherein themolecule specifically binds HBcAg and/or HBeAg. Another way ofdescribing the molecules of this class is by the formula: “X¹ _(n)CZASX²_(n)”, wherein: “X¹” and “X²” are any amino acid and “n” is any integer,“C” is cysteine, “Z” is lysine or arginine”, “A” is alanine, and “S” isserine. In some embodiments, the “X¹ _(n)” or “X² _(n)” encodes anepitope that binds a high titer antibody (e.g., an epitope on a pathogenor a toxin). Other embodiments include an isolated or purified peptidethat is less than 50 amino acids in length having the formula: X¹_(n)CRASX² _(n), wherein “X¹” and “X²” are any amino acid and “n” is anyinteger, and wherein the molecule specifically binds HBcAg and/or HBeAg.As above, another way of describing the molecules of this class is bythe formula: “X¹ _(n)CZASX² _(n)”, wherein: “X¹” and “X²” are any aminoacid and “n” is any integer, “C” is cysteine, “Z” is lysine orarginine”, “A” is alanine, and “S” is serine. In some embodiments, “X¹_(n)” or “X² _(n)” encodes an epitope that binds a high titer antibody.Additional embodiments include a nucleic acid encoding a peptideselected from the group consisting of SEQ. ID. Nos. 1, 2, 3, 45, 54, and74.

[0011] Some embodiments include a method of making a binding partnerthat interacts with HBcAg or HBeAg. By one approach, a region of apolypeptide that interacts with HBcAg or HBeAg is identified, thesequence of said region of the polypeptide is determined, and asynthetic or recombinant binding partner that corresponds to thesequence of said region of the polypeptide is produced. In some aspectsof this embodiment, the polypeptide is an antibody and, in otheraspects, the binding partner is a specificity exchanger. The specificityexchangers described herein are bi-functional binding partners that haveone domain (“specificity domain”), which binds to HBeAg, HBcAg, an HBVviral capsid, or HBV itself, joined to a second domain (“antigenicdomain”) that is recognized by an antibody present in a subject (e.g.,an antigenic domain comprising at least one of the sequences of SEQ. ID.Nos. 79-95). Preferred specificity exchangers include SEQ. ID. Nos: 5,6, 13, 16, 17, 28, 29, 33, 36, 37, and 74.

[0012] Embodiments also include biological complexes comprising thebinding partners described herein, including specificity exchangers,joined to HBeAg, HBcAg, an HBV viral capsid, HBV itself, or fragmentsthereof. Preferred embodiments include complexes comprising the bindingpartners described herein joined to fragments of HBcAG includingpeptides comprising, consisting essentially of, or consisting of thepeptides of SEQ. ID. Nos: 103 and 104 or nucleic acids encoding thesemolecules. These peptides and nucleic acids can be used as researchtools or components for vaccines.

[0013] More embodiments include methods of making a pharmaceutical. Byone approach, a binding partner that interacts with HBcAg or HBeAg isidentified and a therapeutically effective amount of said bindingpartner is incorporated into a pharmaceutical. In preferred aspects ofthis method, the binding partner has a sequence selected from the groupconsisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74. Anothermethod described herein concerns an approach to treat or prevent HBVinfection. Accordingly, a subject in need of a molecule that inhibitsHBV infection is identified and said subject is provided a bindingpartner that interacts with HBcAg or HBeAg, or both. Preferred aspectsof this method involve a binding partner that has a sequence selectedfrom the group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and74.

[0014] Methods of identifying a binding partner that interacts withHBcAg or HBeAg are also embodiments. By one approach, a supportcomprising HBcAg or HBeAg is provided, the support is contacted with acandidate binding partner, and a biological complex comprising HBcAg orHBeAg and the candidate binding partner is detected, wherein detectionof such complex indicates that said candidate binding partner is abinding partner interacts with HBcAg or HBeAg. In preferred aspects ofthis embodiment, the candidate binding partner has an amino acidsequence selected from the group consisting of SEQ. ID. Nos. 1-78.Another method of identifying a binding partner that inhibits HBVinfection involves providing a cell that is infected with HBV,contacting said cell with a candidate binding partner, and identifyingsaid binding partner when the presence of said candidate binding partnerwith said cell is associated with a decrease in HBV infection.

[0015] Furthermore, methods are provided that identify a binding partnerthat modulates an immune system response. Accordingly, one method ispracticed by providing a native antigen presenting cell, contacting saidnaive antigen presenting cell with a binding partner and a T cell thatreacts to HBcAg or HBeAg, and detecting an inhibition or enhancement ofT cell stimulation. In some embodiments, the detection step is performedby evaluating a change in cytokine production or T cell proliferation.

[0016] In another embodiment, a computerized system for identifying abinding partner that interacts with HBcAg or HBeAg is provided. Thissystem includes a first data base comprising protein models of HBcAg orHBeAg; a second data base comprising the composition of a plurality ofcandidate binding partners; a search program that compares the proteinmodel of the first data base with the compositions of the candidatebinding partners of the second database; and a retrieval program thatidentifies a binding partner that interacts with the protein model ofthe first database. In some aspects of this embodiment, the candidatebinding partners have an amino acid sequence selected from the groupconsisting of SEQ. ID. Nos. 1-78.

[0017] Additionally, a computer-based system for identifying a candidatebinding partner having homology to a binding partner is provided. Thissystem has a database with at least one of the sequences of SEQ ID NOS:1-78 or a representative fragment thereof, a search program thatcompares a sequence of a candidate binding partner to sequences in thedatabase to identify homologous sequence(s), and a retrieval programthat obtains said homologous sequence(s).

[0018] A method of determining the presence of HBV in a biologicalsample is also an embodiment. This method is practiced by providing abiological sample, providing a binding partner that binds to HBcAgand/or HBeAg, wherein said binding partner has a sequence selected fromthe group consisting of SEQ. ID. Nos. 4-45, 53, 54, 66-69, 71, and 74,and determining the presence of HBV in the biological sample bymonitoring whether said binding partner binds to HBcAg and/or HBeAg.Diagnostic kits for the detection of HBV infection are embodiments, aswell. One such kit has a binding partner, wherein said binding partnerhas a sequence selected from the group consisting of SEQ. ID. Nos. 4-45,53, 54, 66-69, 71, and 74.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The disclosure herein describes the manufacture,characterization, and use of molecules that bind hepatitis B virus (HBV)core (HBcAg) and e (HBeAg) antigens and thereby inhibit HBV infectionand/or modulate a host immune system response. The molecules that bindto HBcAg and/or HBeAg, such as peptides, modified or derivatizedpeptides, peptidomimetics, and chemicals, are collectively referred toas “binding partners”. Binding partners can be obtained by synthesizingthe heavy (VH) and light (VL) chains of antibodies (e.g., polyclonal,monoclonal, or fragments thereof), synthesizing the domains of proteinsthat interact with HBcAg and/or HBeAg, and by employing techniques inrational drug design and combinatorial chemistry.

[0020] Several synthetic peptides, derived from the variable domains ofmonoclonal antibodies (mAbs) specific for the hepatitis B virus HBcAgand/or HBeAg, were obtained as follows. The mRNAs encoding the VH and VLchains of HBcAg and/or HBeAg monoclonal antibodies (mAbs) were sequencedand the protein sequences corresponding to these mRNAs were determined.Several synthetic peptides corresponding to these sequences were thensynthesized using conventional protein chemistry. These “candidatebinding partners”, which have the potential to bind HBcAg and/or HBeAg,were tested for the ability to interact with HBcAg and HBeAg. Fivepeptides, in particular, were discovered to bind HBcAg and/or HBeAg withhigh affinity and these “high affinity” binding partners had either theconserved motif “CKAS” (SEQ. ID. No. 77) or “CRAS” (SEQ. ID. No. 78).Thus, preferred embodiments include peptides, derivative or modifiedpeptides, or peptidomimetics having the formula “X¹ _(n)CKASX² _(n)” or“X¹ _(n)nCRASX² _(n)” wherein “X¹” and “X²” are any amino acid and “n”is an integer, that bind HBcAg and/or HBeAg. Another way of describingthe molecules of this class is by the formula: “X¹ _(n)CZASX² _(N)”,wherein: “X¹” and “X²” are any amino acid and “n” is any integer, “C” iscysteine, “Z” is lysine or arginine”, “A” is alanine, and “S” is serine.

[0021] By a similar approach, synthetic peptides corresponding to thebinding domains of polyclonal antibodies specific for HBcAg and/or HBeAgcan be manufactured. Polyclonal antibodies specific for HBcAg and/orHBeAg are generated by inoculating animals with HBcAg and/or HBeAg. ThemRNAs encoding the polyclonal antibodies are isolated, sequenced, andthe protein sequences corresponding to these mRNAs are determined.Synthetic peptides corresponding to these protein sequences are thenmade using conventional techniques in protein chemistry. Severalstrategies for obtaining the mRNAs that encode polyclonal antibodiesthat bind HBcAg and/or HBeAg are contemplated including, but not limitedto, yeast one-hybrid screens, yeast two-hybrid screens, and phagedisplay techniques. Ideally, cDNA expression libraries corresponding tomRNAs encoding polyclonal antibodies that bind HBcAg and/or HBeAg arecreated. Embodiments that employ such libraries can express recombinantbinding partners, which can be isolated or purified, characterized, andused in lieu of or in addition to synthetic binding partners.

[0022] The term “isolated” requires that the material be removed fromits original environment (e.g., the natural environment if it isnaturally occurring). For example, a naturally-occurring polypeptidepresent in a living animal is not isolated, but the same polypeptide,separated from some or all of the coexisting materials in the naturalsystem, is isolated. It is also advantageous that the sequences be inpurified form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition. Isolated proteins havebeen conventionally purified to electrophoretic homogeneity by Coomassiestaining, for example. Purification of starting material or naturalmaterial to at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated.

[0023] In addition to antibodies and peptides derived from antibodies,other molecules that bind HBcAg and/or HBeAg can be identified by themethods described herein. That is, techniques in high throughputscreening, combinatorial chemistry, and rational drug design can beemployed to identify more binding partners. By one approach, forexample, a high throughput screen based on a yeast two hybrid system isemployed.

[0024] Accordingly, cDNA expression libraries are generated from anyorganism (e.g., plant, bacteria, virus, insect, amphibian, reptile,bird, or mammal) or a plurality of such organisms or from random ordirected oligonucleotide synthesis. In some embodiments, the organismwill have been immunized for HBcAg and/or HBeAg prior to creation of thecDNA expression library. To create the cDNA library for the yeasttwo-hybrid screen, isolated cDNA made from total mRNA obtained from theorganism or generated by oligonucleotide synthesis is cloned into afirst expression construct having a nucleic acid encoding atranscriptional activation domain (e.g., GAL4). As one of skill willappreciate, the cloning of the first expression construct is conductedsuch that cells having the construct will express a fusion proteincomprising the cDNA of interest and the transcriptional activationdomain when induced.

[0025] Next, a second expression construct (referred to as the “bait”)is made. This construct has nucleic acid encoding HBcAg and/or HBeAg orfragments thereof joined to a transcriptional binding domain (e.g.,GAL4). When a cell having the second expression construct is properlyinduced, a second fusion protein comprising the HBcAg and/or HBeAg orfragments thereofjoined to the transcriptional binding domain (the“bait”) is expressed. The two expression constructs are transferred intoyeast, which harbor a DNA template having at least one DNA bindingdomain specific for the transcriptional binding domain encoded by thebait construct, a minimal promoter, and a downstream reporter gene(e.g., Lac Z or Green Fluorescent Protein (GFP)). When a peptide fromthe cDNA library (i.e., the fusion protein expressed from the firstconstruct) binds to the bait (i.e., the fusion protein from the secondconstruct) a detectable signal is generated from the reporter gene. Theyeast clones can be presented in addressable arrays, which allows forthe precise determination of the clone containing an insert that encodesa protein that binds HBcAg and/or HBeAg. In this manner, protein/proteininteractions between HBcAg and/or HBeAg and the proteins expressed fromthe cDNA library can be rapidly identified.

[0026] Clones that display a signal after induction of the first andsecond constructs but fail to produce a signal without induction areisolated, amplified, and the cDNA inserts are sequenced. The nucleicacid sequence information can be converted to an amino acid sequence andpeptides corresponding to these sequences can be synthesized byconventional protein chemistry. Alternatively, recombinant peptidesexpressed from the positive clones can be isolated and/or purified.These candidate binding partners can then be screened for the ability tointeract with HBcAg and/or HBeAg. The binding partners identified by theapproaches above can also be used as templates for the design ofmodified or derivative peptides, peptidomimetics, and for rational drugdesign. For example, computer modeling and combinatorial chemistry canbe employed to design and manufacture derivative binding partners.

[0027] The term “binding partner” also refers to a bi-functional bindingpartner or “specificity exchanger” comprising a “specificity domain”,which binds HBcAg and/or HBeAg, and an “antigenic domain”, which bindsan antibody or other molecule that can be unrelated to a molecule thatbinds HBcAg and/or HBeAg. Desirable antigenic domains have an epitopefound on a pathogen such as a bacteria, virus, fungi, or mold or atoxin. Preferred antigenic domains have epitopes that bind high titerantibodies (e.g., the antigenic domains described in SEQ. ID. Nos.79-85). These bi-functional binding partners can redirect antibodiesthat already exist in an organism to a desired antigen. Such specifictyexchangers can be manufactured by joining a molecule that binds HBcAgand/or HBeAg, such as a binding partner identified by a method describedabove, to an epitope for any antibody using conventional techniques inmolecular biology. In one embodiment, for example, a specificity domaincomprising the CKAS (SEQ. ID. No. 77) motif was joined to an antigenicdomain comprising the epitope for a monoclonal antibody specific for theherpes simplex virus type 1 gG2 (HSVgG2) protein.

[0028] Desirably, the binding partners are evaluated in a“characterization assay”, which determines the ability of the moleculeto interact with HBcAg and/or HBeAg, inhibit HBV infectivity, ormodulate (inhibit or enhance) a host immune system response. Severalcharacterization assays described herein involve binding assays thatanalyze whether a binding partner can interact with HBcAg and/or HBeAgand to what extent a binding partner can compete with other ligands forHBcAg and/or HBeAg (e.g., multimeric support-based assays and computergenerated binding assays). Additionally, some characterization assaysdetermine the efficacy of binding partners as inhibitors of HBVinfection in vitro and in vivo. Further, characterization assays aredesigned to analyze whether a binding partner can modulate a host immunesystem response, as indicated by the activation of an antigen presentingcell (e.g., a B cell or dendritic cell), production of a cytokine, or Tcell proliferation.

[0029] The binding partners can be used as biotechnological tools,diagnostic reagents, and the active ingredients in pharmaceuticals. Insome embodiments, for example, the binding partners are used asdetection reagents in conventional immunohistochemical techniques. Inother embodiments, the binding partners are expressed in a cell in vitroor in vivo. Still in other embodiments, the binding partners are used asdiagnostic reagents to detect the presence or absence of HBV in abiological sample obtained from a subject. According to this lateraspect, the binding partners can also be used to determine the efficacyof an HBV treatment protocol by monitoring the levels of HBcAg and/orHBeAg before, during, and after treatment.

[0030] Further, binding partners can be incorporated intopharmaceuticals that can be administered to subjects in need of an agentthat interacts with HBcAg and/or HBeAg, such as a human in need oftreatment and/or prevention of HBV infection. Preferably, thesepharmaceuticals comprise formulations having a specificity exchangerthat promotes rapid clearance of HBV particles. Additionally, thepharmaceuticals can include nucleic acid constructs manufactured suchthat binding partners (preferably specificity exchangers) are expressedin a variety of cells of the body. The pharmaceuticals can beadministered to individuals in need of treatment and/or prevention ofHBV infection. The section below describes several approaches toidentify and manufacture binding partners specific for HBcAg and/orHBeAg.

[0031] Identification and Manufacture of Binding Partners Specific forHBCAg and/or HBeAg

[0032] In general, the approach to make the binding partners describedherein involves: (1) obtaining molecules that bind to HBcAg and/orHBeAg; (2) determining the molecular structure or sequence of saidmolecules; and (3) synthesizing peptides that have said molecularstructure or sequence. In one aspect, for example, antibodies or otherpeptides that bind to HBcAg and/or HBeAg are generated and/oridentified; the MRNA sequence encoding the binding partner is obtained,converted to cDNA, and sequenced; and, from this sequence, peptides aresynthesized. The HBcAg and HBeAg-specific peptides can be modified,derivatized, and can also be used as templates for the design ofpeptidomimetics and rational drug discovery. Through techniques incombinatorial chemistry and rational drug design, many more bindingpartners can be identified. The term “binding partner” refers to amolecule that binds HBcAg and/or HBeAg, and should be distinguished fromthe term “candidate binding partner”, which refers to a molecule thatpotentially binds to HBcAg and/or HBeAg. Desirably, binding partnersinhibit viral infectivity and/or modulate (inhibit or enhance) a hostimmune system response (e.g., antigen presenting cell activation,cytokine production, and/or T cell proliferation).

[0033] By one approach, the design and manufacture of peptides that bindHBcAg and HBeAg involves the manufacture of mAbs directed to HBcAg andHBeAg. Depending on the context, the term “antibodies” can encompasspolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library. Furthermore, the terms“low titer antibody” and “high titer antibody” are also used to refer toan antibody having a low avidity to an antigen and a high avidity to anantigen, respectively. That is, whether a particular antibody is a “lowtiter antibody” or a “high titer antibody” depends on the dilution ofantibody containing sera at which an antigen is no longer detectable inan enzyme immunoassay (e.g., an enzyme immunoassay (EIA) or ELISAassay); 200 ng of target antigen is typically used with a 1:1000dilution of secondary antibody. Thus, a “low titer antibody” generallyno longer detects an antigen at a dilution that is less than 1:10000under the conditions for ELISA described above and a “high titerantibody” is characterized by the ability to detect an antigen at adilution that is greater than or equal to 1:10000.

[0034] For the production of antibodies, whether monoclonal orpolyclonal, various hosts including goats, rabbits, rats, mice, etc. canbe immunized by injection with HBcAg and/or HBeAg or any portion,fragment or oligopeptide that retains immunogenic properties. Dependingon the host species, various adjuvants can be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(Bacillus Calmette-Guerin) and Corynebacterium parvum are alsopotentially useful adjuvants.

[0035] Peptides used to induce specific antibodies can have an aminoacid sequence consisting of at least three amino acids, and preferablyat least 10 to 15 amino acids. Short stretches of amino acids encodingfragments of HBcAg and/or HBeAg can be fused with those of anotherprotein such as keyhole limpet hemocyanin such that an antibody isproduced against the chimeric molecule. While antibodies capable ofspecifically recognizing HBcAg and/or HBeAg can be generated byinjecting synthetic 3-mer, 10-mer, and 15-mer peptides that correspondto a protein sequence of a binding partner into an appropriate organism,a more diverse set of antibodies are generated by using recombinantHBcAg and/or HBeAg.

[0036] Monoclonal antibodies directed to HBcAg and/or HBeAg can beprepared using any technique that provides for the production ofantibody molecules by continuous cell lines in culture. These include,but are not limited to, the hybridoma technique originally described byKoehler and Milstein (Nature 256:495-497 (1975)), the human B-cellhybridoma technique (Kosbor et al. Immunol Today 4:72 (1983); Cote et alProc Natl Acad Sci 80:2026-2030 (1983), and the EBV-hybridoma techniqueCole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc,New York N.Y., pp 77-96 (1985)). In addition, techniques developed forthe production of “chimeric antibodies”, the splicing of mouse antibodygenes to human antibody genes to obtain a molecule with appropriateantigen specificity and biological activity can be used. (Morrison etal. Proc Natl Acad Sci 81:6851-6855 (1984); Neuberger et al. Nature312:604-608(1984); Takeda et al. Nature 314:452-454(1985)).Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce HBcAgand/or HBeAg-specific single chain antibodies. Antibodies can also beproduced by inducing in vivo production in the lymphocyte population orby screening recombinant immunoglobulin libraries or panels of highlyspecific binding reagents as disclosed in Orlandi et al., Proc Natl AcadSci 86: 3833-3837 (1989), and Winter G. and Milstein C; Nature349:293-299 (1991).

[0037] Antibody fragments that contain specific binding sites for HBcAgand/or HBeAg can also be generated. For example, such fragments include,but are not limited to, the F(ab′)₂ fragments that can be produced bypepsin digestion of the antibody molecule and the Fab fragments that canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).

[0038] By one approach, monoclonal antibodies to HBcAg and/or HBeAg orfragments thereof are made as follows. Briefly, a mouse is repetitivelyinoculated with a few micrograms of the selected protein or peptidesderived therefrom over a period of a few weeks. The mouse is thensacrificed, and the antibody producing cells of the spleen isolated. Thespleen cells are fused in the presence of polyethylene glycol with mousemyeloma cells, and the excess unfused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution areplaced in wells of a microtiter plate where growth of the culture iscontinued. Antibody-producing clones are identified by detection ofantibody in the supernatant fluid of the wells by immunoassayprocedures, such as ELISA, as originally described by Engvall, E., Meth.Enzymol. 70:419 (1980), and derivative methods thereof. Selectedpositive clones can be expanded and their monoclonal antibody productharvested for use. Detailed procedures for monoclonal antibodyproduction are described in Davis, L. et al. Basic Methods in MolecularBiology Elsevier, New York. Section 21-2. By using the approachdescribed in Example 1, several monoclonal antibodies specific for HBcAgand/or HBeAg were made.

EXAMPLE 1

[0039] The mAbs that were used to create the binding partners specificfor HBcAg and/or HBeAg were made by conventional techniques, asdescribed above, using recombinant peptides. Full length recombinantHBcAg (rHBcAg) encompassing residues 1-183 was produced in Escherichiacoli, as previously described. (Schodel et al., J Biol Chem, 268:1332-7(1993), herein expressly incorporated by reference in its entirety). Atruncated recombinant form of HBeAg containing nine residues of pre-coreand the 150 first residues of HBcAg was also made. Further, anon-structural 3 protein (NS3) of the hepatitis C virus (HCV) was alsomade to serve as a control. (Jin and Peterson, Arch. Biochem. Biophys.,323:47-53 (1995), herein expressly incorporated by reference in itsentirety). Another control peptide, an analogue of HBcAg (AHBcAg),wherein region 76-85 was replaced by an irrelevant sequence, was alsomade.

[0040] Balb/c and CBA mice (purchased from BK Universal, Sollentuna,Sweden) were immunized (Freunds complete adjuvant; CFA) and boosted(Freunds incomplete adjuvant; IFA) by 10 μg of the recombinant peptides.The mice were immunized one to three times, with two weeks between eachimmunization. Three days after the last injection, spleen cells wereharvested and fused with the SP2/0 myeloma cells by standard procedures.The SP2/0 cell lines expressing mAbs were maintained in RPMI-1640 mediumsupplemented with 10% FCS, 2 mM L-Glutamine, 100 U/ml Penicilin and 100μg/ml Streptomycin (GIBCO-BRL, Gaithesburgh, Md.). All cells wereincubated at 37° C. with 7% CO₂. Following three cycles of cloning andscreening by enzyme immunoassay (EIA) using the indicated antigens,stable hybridomas were selected for antibody analysis and extraction ofmRNA. The antibodies were purified on immobilized protein A/G (Pierce,Rockford, Ill.). By using the approach described in this example,several mAbs that bind HBcAg and/or HBeAg were obtained.

[0041] Four of the mAbs obtained by the approach described in Example 1were characterized for their reactivity to HBcAg, ΔHBcAg, denaturatedHBcAg, and HBeAg. The example below describes the approach that was usedto characterize the reactivity of mAbs directed to HBcAg and/or HBeAg.

EXAMPLE 2

[0042] To determine the reactivity and specificity of a mAb, recombinantproteins or fragments thereof (e.g., HBcAg, HBeAg, ΔHBcAg, denaturedHBcAg, or NS3 proteins) were passively adsorbed at 10 μg/ml to 96 wellmicrotiter plates in 50 mM sodium carbonate buffer, pH 9.6, overnight at4° C. Serial dilutions of mAbs were made in phosphate buffered saline(PBS) containing 2% goat serum (Sigma Chemicals, St Louis, MO), and0.05% Tween 20 (PB S-GT). The various dilutions were then incubated onthe plates for 60 minutes. Bound mAbs were detected either by rabbitanti-mouse IgG (Sigma), or rabbit anti-mouse IgG1, IgG2a, IgG2b or IgG3(Sigma) followed by a peroxidase labeled goat anti-rabbit IgG (Sigma).The plates were developed by incubation with dinitro-phenylene-diamine(Sigma) and the absorbance at 490 nm was determined. Additionally, thereactivity in Abbott anti-HBc and HBe IMX assays was determinedaccording to the manufacturer's instructions. The results of thesestudies are provided in TABLE 1. Further analysis revealed that none ofthe mAbs were reactive in the Sorin anti-HBe assay (Sorin Biomedica,Sluggia, Italy) but the 4-2 mAb was reactive in the Abbott IMX anti-Hbeassay, indicating an unusual specificity. TABLE 1 MAb reactivity toHBcAg, mutant HBcAg (ΔHBcAg), denaturated HBcAg (dHBcAg), and HBeAg*.Reactivity in Dominating Abbott IMX assay for Endpoint titre toindicated antigen specificity anti-HBc or anti-HBe MAb HBcAg ΔHBcAgdHBcAg HBeAg in EIA HBc HBe 3-4 3.125 1 1 15.625 HBeAg − − 4-2 3.125 62515.625 125 dHBcAg − + 5H7 625 0 1 1 HBcAg − − 9C8 78.125 25 25 25 HBcAg− −

[0043] Once the specificity of binding of the mAbs was determined, themRNAs encoding the VH and/or VL domains of three mAbs (i.e., mAbs 4-2,5H7, and 9C8) were sequenced and these mRNA sequences were converted toprotein sequences. The next example describes the method that was usedto determine the protein sequence of a binding domain of an antibodythat interacts with HBcAg and/or HBeAg.

EXAMPLE 3

[0044] To determine the protein sequence of an antibody binding domain,total cellular mRNA was extracted using magnetic beads coated witholigo-dT25 (Dynal A.S, Oslo, Norway). The variable domains of the heavy(VH) and light (VL) chains of mAbs were amplified from cDNA by thePolymerase Chain Reaction (PCR) using the recombinant phage antibodysystem (Pharmacia Biotech, Uppsala, Sweden). The amplified cDNAfragments were directly ligated to the TA cloning vector pCR 2.1(Invitrogen, San Diego, USA) as described. (Zhang et al., Clin. Diagn.Lab. Immunol., 7:58-63 (2000), herein expressly incorporated byreference in its entirety). The DNA sequences were determined by anautomated sequencer (ALF express, Pharmacia, Uppsala, Sweden) asdescribed. (Zhang et al., Clin. Diagn. Lab. Immunol., 7:58-63 (2000),herein expressly incorporated by reference in its entirety). From thecDNA sequence, a corresponding protein sequence was deduced. The proteinsequences deduced from VH cDNA clones of mAbs 4-2 and 9C8 and VL cDNAclone 5H7 are provided in TABLE 2 and in the Sequence Listing (SEQ. ID.Nos. 1-3). The approach described above can be used to determine theprotein sequence of the binding domain of either a monoclonal orpolyclonal antibody. TABLE 2 The deduced VH and/or VL sequences of mAbs4-2, 5H7 and 9C8. 4-2 VHVKLQQSGTEVVKPGASVKLSCKASGYIFTSYDIDWVRQTPEQGLEWIGWIFPGE (SEQ. ID. No. 1)GSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSEDSAVYFCARGDYDYYRRYF DLWGQGTTVTVS 5H7VL DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKY (SEQ. ID. No.2) ASNLESGVPARFSGSGSGTDFTLNIHPVEEBDTATYYCQHSWEIPYTFGGGTKLEIKRADAAPAVSIFPPSSKLG 9C8 VHIQLQQSGAELVKPGASVKISCKASGYSFTGYNMNWVKQSHGKSLEWIGNINPY (SEQ. ID. No. 3)YGSTSYNQKFKGKATLTVDKSSSTAYMQLNSLTSEDSAVYYCARGKGTGFAYWGQGTLVTVSAAKTTPPSVYPLVPV

[0045] Synthetic peptides corresponding to the VH and/or VL sequences ofmAbs 4-2, 5H7, and 9C8 or fragments thereof were then synthesized byconventional techniques in protein chemistry. These synthetic peptidesare referred to as “candidate binding partners” because they aremolecules that potentially bind HBcAg and/or HBeAg. The example belowdescribes an approach that was used to synthesize a peptide thatcorresponds to the binding domain of a mAb specific for HBcAg and/orHBeAg.

EXAMPLE 4

[0046] Peptides that correspond to regions of a binding domain of a mAbspecific for HBcAg and/or HBeAg were manufactured as follows.Overlapping peptides (20 amino acids long with a 10 amino acid residueoverlap) corresponding to the VH and/or VL of the mAbs 4-2, 5H7, and 9C8were produced by standard techniques (Sällberg et al., Immunol Lett,30:59-68 (1991), herein expressly incorporated by reference in itsentirety) using a multiple peptide synthesizer and standard Fmocchemistry (Syro, MultiSynTech, Germany). Additional deletion and alaninesubstitution analogues of reactive peptides were synthesized by the sametechnique. In some cases the peptides were purified by high performanceliquid chromatography using standard protocols. (Sallberg et al.,Immunol Lett, 30:59-68 (1991), herein expressly incorporated byreference in its entirety). There are many ways to synthesize or producethe peptides described herein (e.g., through recombinant technology) andthe approach described above is one such method.

[0047] In addition to using monoclonal antibodies, the design andmanufacture of peptides that bind HBcAg and HBeAg can involve themanufacture of polyclonal antibodies directed to HBcAg and HBeAg.Accordingly, animals are repetitively inoculated with HBcAg and/or HBeAgso as to raise a population of high titer polyclonal antibodies specificfor HBcAg and/or HBeAg. The mRNAs that encode the polyclonal antibodiesare isolated, converted to cDNA, and the protein sequences correspondingto these cDNAs are deduced. (See Examples 3 and 4 for an approach tomanufacture peptides that correspond to a binding domain of anantibody). Synthetic peptides having these protein sequences are thenmade using conventional techniques in peptide chemistry.

[0048] Polyclonal antiserum containing antibodies to heterogenousepitopes of a single protein can be prepared by immunizing suitableanimals with HBcAg and/or HBeAg or fragments thereof, which can beunmodified or modified to enhance immunogenicity. Effective polyclonalantibody production is affected by many factors related both to theantigen and the host species. For example, small molecules tend to beless immunogenic than others and can require the use of carriers andadjuvant. Also, host animals vary in response to site of inoculationsand dose, with both inadequate or excessive doses of antigen resultingin low titer antisera. Small doses (ng level) of antigen administered atmultiple intradermal sites appears to be most reliable. An effectiveimmunization protocol for rabbits can be found in Vaitukaitis, J. et al.J. Clin. Endocrinol. Metab. 33:988-991 (1971), herein expresslyincorporated by reference in its entirety.

[0049] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. (See e.g.,Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental ImmunologyD. Wier (ed) Blackwell (1973), herein expressly incorporated byreference in its entirety). Plateau concentration of antibody is usuallyin the range of 0.1 to 0.2 mg/ml of serum (about 12 μM). Affinity of theantisera for the antigen is determined by preparing competitive bindingcurves, as described, for example, by Fisher, D., Chap. 42 in: Manual ofClinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. ForMicrobiol., Washington, D.C. (1980), herein expressly incoprporated byreference in its entirety.

[0050] Once it can be verified that polyclonal antibodies directed toHBcAg and/or HBeAg have been generated, the mRNA obtained from thespleens of animals inoculated with HBcAg and/or HBeAg can be obtainedand used as a template for the synthesis of cDNA. This cDNA, whichrepresents (among other things) RNA encoding regions of polyclonalantibodies, can be inserted into phage that are engineered such that thecDNA insert is expressed and displayed on the surface of the phage. Thatis, a “phage display” cDNA library can be created from the cDNAcorresponding to the mRNA encoding polyclonal antibodies that bind HBcAgand/or HBeAg. Many phage display kits that are suitable for this testingare commercially available.

[0051] Once the phage display library is obtained, a technique called“panning” is employed to isolate phage having an insert that encodes apeptide that binds HBcAg and/or HBeAg. Accordingly, HBcAg and/or HBeAgor fragments thereof are disposed on a support (e.g., a plate) and arebrought in contact with the phage display library. After a sufficienttime for binding has occurred, unbound phage are removed by successivewashes with a isotonic buffer. Next, a plate having a bacterial lawn isbrought in contact with the phage that remain bound to the plate havingimmobilized HBcAg and/or HBeAg. The two plates are held in position forsufficient time for infection of the bacteria and, after inoculation,the plate is incubated overnight at 37° C. The appearance of clear zoneson the bacterial lawn, indicative of phage proliferation, providesevidence that the phage within the zone contain a cDNA that encodes apeptide that binds HBcAg and/or HBeAg. The DNA from such phage can beisolated, sequenced, and the protein sequence of the binding peptidescan be deduced, as described above. Synthetic peptides can then bemanufactured based on these sequences. Further, the cDNA inserts frompositive binding phage can be subcloned into cDNA expression librariesfor the production of recombinant binding partners.

[0052] Another approach to isolate molecules that bind HBcAg and/orHBeAg takes advantage of techniques developed to analyze protein/proteininteractions. Conventional one and two hybrid systems, for example, canbe readily adapted to identify binding partners. Such approachesinclude:

[0053] (1) the two-hybrid systems (Field & Song, Nature 340:245-246(1989); Chien et al., Proc. Natl. Acad. Sci. USA 88:9578-9582 (1991);and Young K H, Biol. Reprod. 58:302-311 (1998), all of which areexpressly incorporated by reference in their entirety);

[0054] (2) reverse two-hybrid system (Leanna & Hannink, Nucl. Acid Res.24:3341-3347 (1996), herein incorporated by reference in theirentirety);

[0055] (3) repressed transactivator system (Sadowski et al., U.S. Pat.No. 5,885,779), herein incorporated by reference in their entirety);

[0056] (4) phage display (Lowman H B, Annu. Rev. Biophys. Biomol.Struct. 26:401-424 (1997), herein incorporated by reference in theirentirety); and

[0057] (5) GST/HIS pull down assays, mutant operators (Granger et al.,WO 98/01879) and the like (See also Mathis G., Clin. Chem. 41:139-147(1995); Lam K. S. Anticancer Drug Res., 12:145-167 (1997); and Phizickyet al., Microbiol. Rev. 59:94-123 (1995), all of which are expresslyincorporated by reference in their entirety).

[0058] An adaptation of the system described by Chien et al., 1991,Proc. Natl. Acad. Sci. USA, 88:9578-9582, herein incorporated byreference in its entirety, which is commercially available from Clontech(Palo Alto, Calif.) is as follows. Plasmids are constructed that encodetwo hybrid proteins: one plasmid consists of nucleotides encoding theDNA-binding domain of a transcription activator protein fused to anucleotide sequence encoding HBcAg and/or HBeAg and the other plasmidconsists of nucleotides encoding the transcription activator protein'sactivation domain fused to a cDNA encoding a candidate binding partner.The DNA-binding domain/HBcAg and/or HBeAg fusion plasmid and thecandidate binding partner cDNA are transformed into a strain of theyeast Saccharomyces cerevisiae that contains a reporter gene (e.g., GFPor lacZ) whose regulatory region contains the transcription activator'sbinding site. Either hybrid protein alone cannot activate transcriptionof the reporter gene: the DNA-binding domain hybrid cannot because itdoes not provide activation function and the activation domain hybridcannot because it cannot localize to the activator's binding sites.Interaction of the two hybrid proteins reconstitutes the functionalactivator protein and results in expression of the reporter gene, whichis detected by an assay for the reporter gene product.

[0059] The two-hybrid system or related methodology can also be used torapidly screen candidate binding partner libraries generated fromanimals for proteins that interact with the “bait” gene product (HBcAgand/or HBeAg/DNA binding domain). For example, total cDNA (representingtotal MRNA) generated from animals that may or may not have polyclonalantibodies specific for HBcAg and/or HBeAg can be fused to the DNAencoding an activation domain. This library and a plasmid encoding ahybrid of a bait gene encoding the HBcAg and/or HBeAg product fused tothe DNA-binding domain are co-transformed into a yeast reporter strain,and the resulting transformants are screened for those that express thereporter gene.

[0060] For example, and not by way of limitation, a bait gene sequenceencoding HBcAg and/or HBeAg can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein. These colonies are purified and the library plasmidsresponsible for reporter gene expression are isolated. DNA sequencing isthen used to identify the proteins encoded by the library plasmids.

[0061] A cDNA/activation domain library representative of the total mRNAfrom an organism that may or may not have polyclonal antibodies directedto HBcAg and/or HBeAg and an activation domain that interacts with theHBcAg and/or HBeAg/DNA binding domain fusion protein can be made usingmethods routinely practiced in the art. According to the particularsystem described herein, for example, the cDNA fragments can be insertedinto a vector such that they are translationally fused to thetranscriptional activation domain of GAL4. This library can beco-transformed along with the bait HBcAg and/or HBeAg gene-GAL4 fusionplasmid into a yeast strain that contains a LacZ gene driven by apromoter which contains GAL4 activation sequence. A cDNA encodedprotein, fused to GAL4 transcriptional activation domain, that interactswith bait HBcAg and/or HBeAg gene product will reconstitute an activeGAL4 protein and thereby drive expression of the LacZ gene. Coloniesthat express lacZ can be detected and the cDNA can then be purified fromthese strains, sequenced, and synthetic peptides corresponding to thesesequences can be generated, derivatized modified, or used as templatesfor rational drug design. The section below describes binding partnersin greater detail.

[0062] Binding Partners Specific for HBcAg and/or HBeAg

[0063] While the most preferred peptide embodiments are at least 13amino acids in length, many candidate binding partners and bindingpartners are between about 3 amino acids and about 100 amino acids ormore in length. That is, desirable candidate binding partners andbinding partners are about 3-125 amino acids in length, more desirablecandidate binding partners and binding partners are between about 3-100amino acids in length, preferred candidate binding partners and bindingpartners are between about 3-75 amino acids in length, more preferredcandidate binding partners and binding partners are between about 3-50amino acids in length, and most preferred candidate binding partners andbinding partners are between about 13-25 amino acids in length. Someembodiments, for example have the formula “X¹ _(n)CKASX² _(n))” or “X¹_(n)CRASX² _(n)”, wherein “X¹” and “X²” are any amino acid and “n” isany integer, and the molecule specifically binds HBcAg and/or HBeAg.Another way of describing the molecules of this class is by the formula:“X¹ _(n)CZASX² _(n)”, wherein: “X¹” and “X²” are any amino acid and “n”is any integer, “C” is cysteine, “Z” is lysine or arginine”, “A” isalanine, and “S” is serine.

[0064] The peptides not only include those molecules containing as aprimary amino acid sequence all or part of the amino acid sequence ofSEQ. ID. Nos.1-78, for example, but also altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a silent change. Accordingly, one ormore amino acid residues within the sequence of SEQ. ID. Nos. 1-78 canbe substituted by another amino acid of a similar polarity that acts asa functional equivalent, resulting in a silent alteration. Substitutesfor an amino acid within the sequence can be selected from other membersof the class to which the amino acid belongs. For example, the non-polar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. The uncharged polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine, and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Thearomatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0065] Additional embodiments are nucleic acids that encode the bindingpartners and candidate binding partners described herein. The nucleicacid embodiments can be DNA or RNA and these molecules can be providedin constructs, plasmids, vectors, chromosomes and can be transferred toplants, virus, bacteria, insects, amphibians, reptiles, birds, animals,and mammals, including humans. Most preferably, the nucleic acidembodiments are between about 9 and about 300 nucleotides in length,although it is recognized that a fusion protein of almost any length canincorporate a nucleic acid embodiment. That is, the nucleic acidembodiments desirably have about 9-1000 nucleotides, preferably about9-700 nucleotides, more preferably about 9-500 nucleotides, and mostpreferably about 9-300 nucleotides. Some nucleic acid embodiments, forexample encode a peptide having the formula “X¹ _(n)CKASX² _(n)” or “X¹_(n)CRASX² _(n)”, wherein “X¹” and “X²” are any amino acid and “n” isany integer and wherein the peptide encoded by the nucleic acidspecifically binds to HBcAg and/or HBeAg. The section below describesthe manufacture and use of modified and derivatized binding partnersthat resemble peptides (e.g., peptidomimetics) that bind HbcAg and/orHBeAg.

[0066] Modified and Derivatized Binding Partners Specific for HBcAgand/or HBeAg

[0067] The peptides described herein can be modified (e.g., the bindingpartners can have substituents not normally found on a peptide or thebinding partners can have substituents that are normally found on thepeptide but are incorporated at regions of the peptide that are notnormal). The peptides can be acetylated, acylated, or aminated, forexample. Substituents that can be included on the peptide so as tomodify it include, but are not limited to, H, alkyl, aryl, alkenyl,alkynl, aromatic, ether, ester, unsubstituted or substituted amine,amide, halogen or unsubstituted or substituted sulfonyl or a 5 or 6member aliphatic or aromatic ring. Thus, the term “binding partner” canrefer to a modified or unmodified peptide and a chemical or apeptidomimetic that structurally (three-dimensionally ortwo-dimensionally) resembles a peptide that binds HBcAg and/or HBeAg.

[0068] There are many ways to make a peptidomimetic that resembles thepeptides described herein. The naturally occurring amino acids employedin the biological production of peptides all have the L-configuration.Synthetic peptides can be prepared employing conventional syntheticmethods, utilizing L-amino acids, D-amino acids, or various combinationsof amino acids of the two different configurations. Synthetic compoundsthat mimic the conformation and desirable features of a binding partnerbut that avoid the undesirable features, e.g., flexibility (loss ofconformation) and bond breakdown are known as a “peptidomimetics”. (See,e.g., Spatola, A. F. Chemistry and Biochemistry of Amino Acids.Peptides, and Proteins (Weistein, B, Ed.), Vol. 7, pp. 267-357, MarcelDekker, New York (1983), which describes the use of the methylenethiobioisostere [CH₂ S] as an amide replacement in enkephalin analogues; andSzelke et al., In peptides: Structure and Function, Proceedings of theEighth American Peptide Symposium, (Hruby and Rich, Eds.); pp. 579-582,Pierce Chemical Co., Rockford, Ill. (1983), which describes renininhibitors having both the methyleneamino [CH₂ NH] and hydroxyethylene[CHOHCH₂ ] bioisosteres at the Leu-Val amide bond in the 6-13octapeptide derived from angiotensinogen, all of which are expresslyincorporated by reference in their entireties).

[0069] In general, the design and synthesis of a peptidomimetic involvesstarting with the sequence of the peptide and the conformation data(e.g., geometry data, such as bond lengths and angles) of a desiredpeptide (e.g., the most probable simulated peptide), and using such datato determine the geometries that should be designed into thepeptidomimetic. Numerous methods and techniques are known in the art forperforming this step, any of which could be used. (See, e.g., Farmer, P.S., Drug Design, (Ariens, E. J. ed.), Vol. 10, pp. 119-143 (AcademicPress, New York, London, Toronto, Sydney and San Francisco) (1980);Farmer, et al., in TIPS, 9/82, pp. 362-365; Verber et al., in TINS,9/85, pp. 392-396; Kaltenbronn et al., in J. Med. Chem. 33: 838-845(1990); and Spatola, A. F., in Chemistry and Biochemistry of AminoAcids. Peptides, and Proteins, Vol. 7, pp. 267-357, Chapter 5, “PeptideBackbone Modifications: A Structure-Activity Analysis of PeptidesContaining Amide Bond Surrogates. Conformational Constraints, andRelations” (B. Weisten, ed.; Marcell Dekker: New York, pub.) (1983);Kemp, D. S., “Peptidomimetics and the Template Approach to Nucleation ofβ-sheets and α-helices in Peptides,” Tibech, Vol. 8, pp. 249-255 (1990),all of which are expressly incorporated by reference in theirentireties. Additional teachings can be found in U.S. Pat. Nos.5,288,707; 5,552,534; 5,811,515; 5,817,626; 5,817,879; 5,821,231; and5,874,529, all of which are expressly incorporated by reference in theirentireties. Once the peptidomimetic is designed, it can be made usingconventional techniques in peptide chemistry and/or organic chemistry.

[0070] Preferred peptidomimetics have structures that resemble a peptidewhose sequence is provided in SEQ. ID. No. 1-78. While the mostpreferred peptidomimetics have structures that resemble peptides thatare at least 13 amino acids in length, many peptidomimetics resemblepeptides that are between about 3 amino acids and about 100 amino acidsor more in length. That is, desirable peptidomimetics resemble peptidesthat are about 3-125 amino acids in length, more desirablepeptidomimetics resemble peptides that are between about 3-100 aminoacids in length, preferred peptidomimetics resemble peptides that arebetween about 3-75 amino acids in length, more preferred peptidomimeticsresemble peptides that are between about 3-50 amino acids in length, andmost preferred peptidomimetics resemble peptides that are between about13-25 amino acids in length. Some embodiments, for example, arepeptidomimetics that resemble a peptide having the formula “X¹_(n)CKASX² _(n)” or “X¹ _(n)CRASX² _(n)”, wherein “X¹” and “X²” are anyamino acid and “n” is any integer, and the molecule specifically bindsHBcAg and/or HBeAg. In the discussion that follows, several methods ofusing candidate binding partners and binding partners as templates formolecular modeling and rational drug design are described. Thesetechniques can be applied to identify additional molecules that bind toHBcAg and/or HBeAg and thereby inhibit viral infectivity and/or modulatea host immune response.

[0071] Rational Drug Design Approaches to Identify Binding PartnersSpecific for HBcAg and/or HBeAg

[0072] Several methods of molecular modeling and rational drug designcan be used to identify more molecules that bind to HBcAg and/or HBeAg.Rational drug design involving polypeptides requires identifying anddefining a first peptide and using this first target peptide to definethe requirements for a second peptide. With such requirements defined,one can find or prepare an appropriate peptide or non-peptide moleculethat meets all or substantially all of the defined requirements. Thus,one goal of rational drug design is to produce structural or functionalanalogs of biologically active polypeptides of interest in order tofashion drugs that are, for example, more or less potent forms of aparticular binding partner. (See, e.g., Hodgson, Bio. Technology 9:19-21(1991)).

[0073] Combinatorial chemistry can also be used to rapidly make and testthe materials constructed by rational drug design. Combinatorialchemistry is the science of synthesizing and testing compounds forbioactivity en masse, instead of one by one, the aim being to discoverdrugs and materials more quickly and inexpensively than was formerlypossible. Many high throughput systems for rapidly testing whether atarget molecule can be bound by a candidate compound are known in theart. These systems can be adapted to determine whether a candidatebinding partner can interact with HBcAg and/or HBeAg or a fragmentthereof.

[0074] Rational drug design and combinatorial chemistry have become moreintimately related in recent years due to the development of approachesin computer-aided protein modeling and drug discovery. (See e.g., U.S.Pat. Nos. 4,908,773; 5,884,230; 5,873,052; 5,331,573; and 5,888,738).Not only is it possible to view molecules on computer screens in threedimensions but it is also possible to examine the interactions ofmacromolecules such as enzymes and receptors and rationally designderivative molecules to test. (See Boorman, Chem. Eng. News 70:18-26(1992)). A vast amount of user-friendly software and hardware is nowavailable and virtually all pharmaceutical companies have computermodeling groups devoted to rational drug design. Molecular SimulationsInc. (www.msi.com), for example, sells several sophisticated programsthat allow a user to start from an amino acid sequence, build a two orthree-dimensional model of the protein or polypeptide, compare it toother two and three-dimensional models, and analyze the interactions ofcompounds, drugs, and peptides with a three dimensional model in realtime.

[0075] Accordingly, in some embodiments, software is used to compareregions of binding partners with other molecules, such as peptides,peptidomimetics, and chemicals, so that therapeutic interactions can bepredicted and designed (See Schneider, Genetic Engineering NewsDecember: page 20 (1998), Tempczyk et al., Molecular Simulations Inc.Solutions April (1997) and Butenhof, Molecular Simulations Inc. CaseNotes (August 1998) for a discussion of molecular modeling). Forexample, the protein or nucleic acid sequence of a candidate bindingpartner, binding partner, or a domain of these molecules can be enteredonto a computer readable medium for recording and manipulation. It willbe appreciated by those skilled in the art that a computer readablemedium having these sequences can interface with software that convertsor manipulates the sequences to obtain structural and functionalinformation, such as protein models. That is, the functionality of asoftware program that converts or manipulates these sequences includesthe ability to compare these sequences to other sequences or structuresof molecules that are present on publicly and commercially availabledatabases so as to conduct rational drug design.

[0076] The candidate binding partner or binding partner polypeptide ornucleic acid sequence or both can be stored, recorded, and manipulatedon any medium that can be read and accessed by a computer. As usedherein, the words “recorded” and “stored” refer to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information on acomputer readable medium to generate manufactures comprising the desirednucleotide or polypeptide sequence information. A variety of datastorage structures are available to a skilled artisan for creating acomputer readable medium having recorded thereon a nucleotide orpolypeptide sequence. The choice of the data storage structure willgenerally be based on the component chosen to access the storedinformation. Computer readable media include magnetically readablemedia, optically readable media, or electronically readable media. Forexample, the computer readable media can be a hard disc, a floppy disc,a magnetic tape, zip disk, CD-ROM, DVD-ROM, RAM, or ROM as well as othertypes of other media known to those skilled in the art. The computerreadable media on which the sequence information is stored can be in apersonal computer, a network, a server or other computer systems knownto those skilled in the art.

[0077] Embodiments utilize computer-based systems that contain thesequence information described herein and convert this information intoother types of usable information (e.g., protein models for rationaldrug design). The term “a computer-based system” refers to the hardware,software, and any database used to analyze a candidate binding partneror a binding partner nucleic acid or polypeptide sequence or both, orfragments of these biomolecules so as to construct models or to conductrational drug design. The computer-based system preferably includes thestorage media described above, and a processor for accessing andmanipulating the sequence data. The hardware of the computer-basedsystems of this embodiment comprise a central processing unit (CPU) anda database. A skilled artisan can readily appreciate that any one of thecurrently available computer-based systems are suitable.

[0078] In one particular embodiment, the computer system includes aprocessor connected to a bus that is connected to a main memory(preferably implemented as RAM) and a variety of secondary storagedevices, such as a hard drive and removable medium storage device. Theremovable medium storage device can represent, for example, a floppydisk drive, a DVD drive, an optical disk drive, a compact disk drive, amagnetic tape drive, etc. A removable storage medium, such as a floppydisk, a compact disk, a magnetic tape, etc. containing control logicand/or data recorded therein can be inserted into the removable storagedevice. The computer system includes appropriate software for readingthe control logic and/or the data from the removable medium storagedevice once inserted in the removable medium storage device. Thecandidate binding partner or binding partner nucleic acid or polypeptidesequence or both can be stored in a well known manner in the mainmemory, any of the secondary storage devices, and/or a removable storagemedium. Software for accessing and processing these sequences (such assearch tools, compare tools, and modeling tools etc.) reside in mainmemory during execution.

[0079] As used herein, “a database” refers to memory that can store acandidate binding partner or binding partner nucleotide or polypeptidesequence information, protein model information, information on otherpeptides, chemicals, peptidomimetics, and other agents that interactwith HbcAg and/or HBeAg, and values or results from characterizationassays. Additionally, a “database” refers to a memory access componentthat can access manufactures having recorded thereon candidate bindingpartner or binding partner nucleotide or polypeptide sequenceinformation, protein model information, information on other peptides,chemicals, peptidomimetics, and other agents that interact with HbcAgand/or HBeAg, and values or results from characterization assays. Thesequence data and values or results from characterization assays can bestored and manipulated in a variety of data processor programs in avariety of formats. For example, the sequence data can be stored as textin a word processing file, such as Microsoft WORD or WORDPERFECT, anASCII file, a html file, or a pdf file in a variety of database programsfamiliar to those of skill in the art, such as DB2, SYBASE, or ORACLE.

[0080] A “search program” refers to one or more programs that areimplemented on the computer-based system to compare a candidate bindingpartner or binding partner nucleotide or polypeptide sequence with othernucleotide or polypeptide sequences and other agents including but notlimited to peptides, peptidornimetics, and chemicals stored within adatabase. A search program also refers to one or more programs thatcompare one or more protein models to several protein models that existin a database and one or more protein models to several peptides,peptidomimetics, and chemicals that exist in a database. Still further,a search program can be used to compare values or results fromcharacterization assays and agents that modulate bindingpartner-mediated effects on viral infectivity and/or host immune systemresponse.

[0081] A “retrieval program” refers to one or more programs that can beimplemented on the computer-based system to identify a homologousnucleic acid sequence, a homologous protein sequence, or a homologousprotein model. A retrieval program can also used to identify peptides,peptidomimetics, and chemicals that interact with HBcAg and/or HBeAg ora protein model of HBcAg and/or HBeAg stored in a database. A retrievalprogram can also be used to obtain “a binding partner profile” that iscomposed of a chemical structure, nucleic acid sequence, or polypeptidesequence or model of an molecule that interacts with HBcAg and/or HBeAgand, thereby inhibits viral infectivity or modulates a host immuneresponse to HBV.

[0082] As a starting point to rational drug design, a two or threedimensional model of a polypeptide of interest is created (e.g., abinding partner or candidate binding partner whose sequence is providedin SEQ. ID. Nos. 1-78). In the past, the three-dimensional structure ofproteins has been determined in a number of ways. Perhaps the best knownway of determining protein structure involves the use of x-raycrystallography. A general review of this technique can be found in VanHolde, K. E. Physical Biochemistry, Prentice-Hall, N.J. pp. 221-239(1971), herein expressly incorporated by reference in its entirety.Using this technique, it is possible to elucidate three-dimensionalstructure with good precision. Additionally, protein structure can bedetermined through the use of techniques of neutron diffraction, or bynuclear magnetic resonance (NMR). (See, e.g., Moore, W. J., PhysicalChemistry, 4^(th) Edition, Prentice-Hall, N.J. (1972), herein expresslyincorporated by reference in its entirety).

[0083] Alternatively, protein models of a polypeptide of interest can beconstructed using computer-based protein modeling techniques. By oneapproach, the protein folding problem is solved by finding targetsequences that are most compatible with profiles representing thestructural environments of the residues in known three- dimensionalprotein structures. (See, e.g., U.S. Pat. No. 5,436,850, hereinexpressly incorporated by reference in its entirety). In anothertechnique, the known three-dimensional structures of proteins in a givenfamily are superimposed to define the structurally conserved regions inthat family. This protein modeling technique also uses the knownthree-dimensional structure of a homologous protein to approximate thestructure of a polypeptide of interest. (See e.g., U.S. Pat. Nos.5,557,535; 5,884,230; and 5,873,052, all of which are expresslyincorporated by reference in their entireties). Conventional homologymodeling techniques have been used routinely to build models ofproteases and antibodies. (Sowdhamini et al., Protein Engineering10:207, 215 (1997), herein expressly incorporated by reference in itsentirety). Comparative approaches can also be used to developthree-dimensional protein models when the protein of interest has poorsequence identity to template proteins. In some cases, proteins foldinto similar three-dimensional structures despite having very weaksequence identities. For example, the three-dimensional structures of anumber of helical cytokines fold in similar three-dimensional topologyin spite of weak sequence homology.

[0084] The recent development of threading methods and “fuzzy”approaches now enables the identification of likely folding patterns andfunctional protein domains in a number of situations where thestructural relatedness between target and template(s) is not detectableat the sequence level. By one method, fold recognition is performedusing Multiple Sequence Threading (MST) and structural equivalences arededuced from the threading output using the distance geometry programDRAGON that constructs a low resolution model. A full-atomrepresentation is then constructed using a molecular modeling packagesuch as QUANTA.

[0085] According to this 3-step approach, candidate templates are firstidentified by using the novel fold recognition algorithm MST, which iscapable of performing simultaneous threading of multiple alignedsequences onto one or more 3-D structures. In a second step, thestructural equivalences obtained from the MST output are converted intointerresidue distance restraints and fed into the distance geometryprogram DRAGON, together with auxiliary information obtained fromsecondary structure predictions. The program combines the restraints inan unbiased manner and rapidly generates a large number of lowresolution model confirmations. In a third step, these low resolutionmodel confirmations are converted into full-atom models and organized toenergy minimization using the molecular modeling package QUANTA. (Seee.g., Aszódi et al., Proteins:Structure, Function, and Genetics,Supplement 1:38-42 (1997), herein expressly incorporated by reference inits entirety).

[0086] In a preferred approach, the commercially available “Insight II98” program (Molecular Simulations Inc.) and accompanying modules areused to create a two and/or three dimensional model of a polypeptide ofinterest from an amino acid sequence. Insight II is a three-dimensionalgraphics program that can interface with several modules that performnumerous structural analysis and enable real-time rational drug designand combinatorial chemistry. Modules such as Builder, Biopolymer,Consensus, and Converter, for example, allow one to rapidly create a twodimensional or three dimensional model of a polypeptide, carbohydrate,nucleic acid, chemical or combinations of the foregoing from theirsequence or structure. The modeling tools associated with Insight IIsupport many different data file formats including Brookhaven andCambridge databases; AMPAC/MOPAC and QCPE programs; Molecular DesignLimited Molfile and SD files, Sybel Mol2 files, VRML, and Pict files.

[0087] Additionally, the techniques described above can be supplementedwith techniques in molecular biology to design models of the protein ofinterest. For example, a known binding partner can be analyzed by analanine scan (Wells, Methods in Enzymol. 202:390-411 (1991), hereinexpressly incorporated by reference in its entirety) or other types ofsite-directed mutagenesis analysis. In alanine scan, each amino acidresidue of the binding partner is sequentially replaced by alanine in astep-wise fashion (i.e., only one alanine point mutation is incorporatedper molecule starting at position #1 and proceeding through the entiremolecule), and the effect of the mutation on the peptide's activity in acharacterization assay is determined. Each of the amino acid residues ofthe peptide is analyzed in this manner and the regions important for thebinding to HBcAg and/or HBeAg are determined. These functionallyimportant regions can be recorded on a computer readable medium, storedin a database in a computer system, and a search program can be employedto generate a protein model of the functionally important regions. Theexample below describes a rational drug design approach that was used toidentify fragments of the binding domains of mAbs that specifically bindHBcAg and/or HBeAg.

EXAMPLE 5

[0088] One approach to rational drug design involves sequential aminoacid deletion of a known binding partner starting from either the aminoor carboxy termini. Amino-terminal deletions of the binding partners ofSEQ. ID. Nos. 5 and 17 were made and these peptide fragments were joinedto a support and analyzed for the ability to bind HBcAg. By using thistechnique, a fine map of the peptide sequence involved in binding toHBcAg was obtained. As shown in TABLE 3, the HBcAg binding sequences forthe peptides of SEQ. ID. Nos. 5 and 17 included KLSCKASGYIFTS (SEQ. ID.No. 45) and CRASQSVSTSSYSYMHWY (SEQ. ID. No. 54), respectively. Theamino-terminal deletions of the peptide of SEQ. ID. Nos. 5 were alsoevaluated for the ability to inhibit binding of mAb 4-2 to HBcAg. (SeeTABLE 4). The amino- terminal deletion products of the peptide of SEQ.ID. No 5 that were most effective at inhibiting binding of mAb 4-2 werefound to have at least the sequence VKLSCKASGYIFTS (SEQ. ID. No. 44),which provided evidence that the valine residue in SEQ. ID. No. 44 wasintimately involved in binding of mAb4-2 to HBcAg. TABLE 3 Mapping ofthe HBcAg binding sequence using support-bound amino terminal deletionpeptides* OD at Amino terminal deletion peptides 490 nm VKPGASVKLSCKASGYIFTS (SEQ. ID. No. 5) 3.257   KPGASVKLSCKASGYIFTS (SEQ.ID. No. 39) 1.337    PGASVKLSCKASGYIFTS (SEQ. ID. No. 40) 1.722    GASVKLSCKASGYIFTS (SEQ. ID. No. 41) 2.863      ASVKLSCKASGYIFTS(SEQ. ID. No. 42) 3.219       SVKLSCKASGYIFTS (SEQ. ID. No. 43) 3.364       VKLSCKASGYIFTS (SEQ. ID. No. 44) 3.703         KLSCKASGYIFTS(SEQ. ID. No. 45) 3.694          LSCKASGYIFTS (SEQ. ID. No. 46) 0.565          SCKASGYIFTS (SEQ. ID. No. 47) 0.297            CKASGYIFTS(SEQ. ID. No. 48) 0.255             KASGYIFTS (SEQ. ID. No. 49) 0.237             ASGYIFTS (SEQ. ID. No. 50) 0.407               SGYIFTS(SEQ. ID. No. 51) 0.389                GYIFTS (SEQ. ID. No. 52) 0.414ISCRASQSVSTSSYSYMHWY (SEQ. ID. NO.17) 1.939   SCRASQSVSTSSYSYMHWY (SEQ.ID. No. 53) 1.452    CRASQSVSTSSYSYMHWY (SEQ. ID. No. 54) 1.415    RASQSVSTSSYSYMHWY (SEQ. ID. No. 55) 0.429      ASQSVSTSSYSYMHWY(SEQ. ID. No. 56) 0.324       SQSVSTSSYSYMHWY (SEQ. ID. No. 57) 0.310       QSVSTSSYSYMHWY (SEQ. ID. No. 58) 0.282         SVSTSSYSYMHWY(SEQ. ID. No. 59) 0.305          VSTSSYSYMHWY (SEQ. ID. No. 60) 0.369          STSSYSYMHWY (SEQ. ID. No. 61) 0.372            TSSYSYMHWY(SEQ. ID. No. 62) 0.317             SSYSYMHWY (SEQ. ID. No. 63) 0.311             SYSYMHWY (SEQ. ID. No. 63) 0.283               YSYMHWY(SEQ. ID. No. 64) 0.245                SYMHWY (SEQ. ID. No. 65) 0.218

[0089] TABLE 4 Inhibition of mAb 4-2 binding to HBcAg by the aminoterminal deletion peptides added prior to addition of mAb*. OD at Aminoterminal deletion peptides 490 nm VKPGASVKLSCKASGYIFTS (SEQ. ID. No. 5)0.453  KPGASVKLSCKASGYIFTS (SEQ. ID. No. 39) 0.202   PGASVKLSCKASGYIFTS(SEQ. ID. No. 40) 0.182    GASVKLSCKASGYIFTS (SEQ. ID. No. 41) 0.205    ASVKLSCKASGYIFTS (SEQ. ID. No. 42) 0.207      SVKLSCKASGYIFTS (SEQ.ID. No. 43) 0.175       VKLSCKASGYIFTS (SEQ. ID. No. 44) 0.152       KLSCKASGYIFTS (SEQ. ID. No. 45) 0.808         LSCKASGYIFTS (SEQ.ID. No. 46) 0.777          SCKASGYTFTS (SEQ. ID. No. 47) 0.784          CKASGYIFTS (SEQ. ID. No. 48) 0.851            KASGYIFTS (SEQ.ID. No. 49) 0.866             ASGYIETS (SEQ. ID. No. 50) 0.920             SGYIFTS (SEQ. ID. No. 51) 0.887               GYIFTS (SEQ.ID. No. 52) 0.903

[0090] Once a model or map of a binding partner is created, it can becompared to other models or maps so as to identify new members of aparticular binding partner family. By starting with the amino acidsequence or- protein model of a binding partner, for example, moleculeshaving two-dimensional and/or three-dimensional homology can be rapidlyidentified. In one approach, a percent sequence identity can bedetermined by standard methods that are commonly used to compare thesimilarity and position of the amino acid of two polypeptides. Using acomputer program such as BLAST or FASTA, two polypeptides can be alignedfor optimal matching of their respective amino acids (either along thefull length of one or both sequences, or along a predetermined portionof one or both sequences). Such programs provide “default” openingpenalty and a “default” gap penalty, and a scoring matrix such as PAM250 (a standard scoring matrix; see Dayhoff et al., in: Atlas of ProteinSequence and Structure, Vol. 5, Supp. 3 (1978)) can be used inconjunction with the computer program. The percent identity can then becalculated as:$\frac{\left( \text{total~~~number~~~of~~~identical~~~matches} \right)}{\begin{matrix}{\text{[length~~~of~~~the~~~longer~~~sequence~~~within~~~the~~~matched~~~span} +} \\\text{number~~~of~~~gaps~~~introduced~~~into~~~the~~~longer~~~sequence} \\\text{in~~~order~~~to~~~align~~~the~~~two~~~sequences]}\end{matrix}} \times 100$

[0091] total number of identical matches X 100 [length of the longersequence within the matched span +number of gaps introduced into thelonger sequence in order to align the two sequences]

[0092] Accordingly, the protein sequence corresponding to a bindingpartner or a binding partner or a fragment or derivative of thesemolecules can be compared to known sequences on a protein basis. Proteinsequences corresponding to a binding partner, or a binding partner or afragment or derivative of these molecules are compared, for example, toknown amino acid sequences found in Swissprot release 35, PIR release 53and Genpept release 108 public databases using BLASTP with the parameterW=8 and allowing a maximum of 10 matches. In addition, the proteinsequences are compared to publicly known amino acid sequences ofSwissprot using BLASTX with the parameter E=0.001. The example belowdescribes database searches that were performed on the identifiedbinding partners so as to find homologous molecules that are expected tobind HBcAg and/or HBeAg.

EXAMPLE 6

[0093] To identify new candidate binding partners, the sequences ofidentified binding partners were used to search publicly availabledatabases. The sequences KLSCKASGYIFTS (SEQ. ID. No. 45) andCRASQSVSTSSYSYMHWY (SEQ. ID. No. 54), obtained from mus musculins, wereused to search for homologous molecules in Genebank, for example. Manysequences with a high degree of homology were found. Noticeably, thesequences uncovered in the search were mAb sequences from variousspecies including, Homo sapiens, Carassius auratus, Canis familiaris,and Caiman crocodilus. Fragments of these sequences are provided inTABLE 5 and the Sequence Listing (SEQ. ID. Nos. 66, 67, 68, and 69). Notonly did these findings demonstrate that homology-based methods ofrational drug design can yield new candidate binding partners but thedata also provided evidence that HBcAg can bind naive B cells in aplurality of different species. TABLE 5 Alignment of an HBcAg and HBeAgbinding with sequences obtained from a Genbank and Swissprot search*Genebank/ swissprot Sequence identity Sequence accession Pept. #2 (musmusc.) V K P G A S V K L S C K A S G Y     F T S (SEQ. ID. No. 5) Homosapiens     P G A S V R I S C K A S G Y P80421     A F (SEQ. ID. No. 66)Carassius auratus   K P G D S L R L S C K A S G Y P19180     T F S (SEQ.ID. No. 67) Canis familiaris V K P G G S L R L S C V A S G F P01785    F S S (SEQ. ID. No. 68) Caiman crocodilus   K P G DS L R L S C K G S G F P03981     F S N (SEQ. ID. No. 69) *The homologysearch was made prior to December 11, 1999.

[0094] In another embodiment, computer modeling and thesequence-to-structure-to-function paradigm is exploited to identify morebinding partners and candidate binding partners. By this approach, firstthe structure of a binding partner or a candidate binding partner havinga known response in a characterization assay is determined from itssequence using a threading algorithm, which aligns the sequence to thebest matching structure in a structural database. Next, the peptide'sactive site (i.e., the site important for a desired response in thecharacterization assay) is identified and a “fuzzy functional form”(FFF)—a three-dimensional descriptor of the active site of a protein—iscreated. (See e.g., Fetrow et al., J. Mol. Biol. 282:703-711 (1998) andFetrow and Skolnick, J. Mol. Biol. 281: 949-968 (1998), herein expresslyincorporated by reference in its entirety). The mapping techniquesdescribed above can be used to facilitate description of the active siteof the peptide.

[0095] The FFFs are built by iteratively superimposing the proteingeometries from a series of functionally related proteins with knownstructures. The FFFs are not overly specific, however, and the degree towhich the descriptors can be relaxed is explored. In essence, conservedand functionally important residues for a desired response areidentified and a set of geometric and conformational constraints for aspecific function are defined in the form of a computer algorithm. Theprogram then searches experimentally determined protein structures froma protein structural database for sets of residues that satisfy thespecified constraints. In this manner, homologous three-dimensionalstructures can be compared and degrees (e.g., percentages ofthree-dimensional homology) can be ascertained. The ability to searchthree-dimensional structure databases for structural similarity to aprotein of interest can also be accomplished by employing the Insight IIusing modules such as Biopolymer, Binding Site Analysis, andProfiles-3D.

[0096] By using this computational protocol, genome sequence data basessuch as maintained by various organizations including:http://www.tigr.org/db; http://www.genetics.wisc.edu;http://genome-www.stanford.edu/˜ball; http://hiv-web.1anl.gov:http://wwwncbi.nlm.nih.gov: http://www.ebi.ac.uk;http://pasteur.fr/other/biology; and http://www.genome.wi.mit.edu, canbe rapidly screened for specific protein active sites and foridentification of the residues at those active sites that resemble adesired molecule. Several other groups have developed databases of shortsequence patterns or motifs designed to identify a given function oractivity of a protein. Many of these databases, notably Prosite(http://expasy.hcuge.ch/sprot/prosite.html): Blocks(http://www.blocks.fhcrc.org); Prints(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html), theMolecular Modelling Database (MMDB), and the Protein Data Bank can useshort stretches of sequence information to identify sequence patternsthat are specific for a given function; thus they avoid the problemsarising from the necessity of matching entire sequences.

[0097] By a similar approach, a binding partner can be identified andmanufactured as follows. First, a molecular model of one or more bindingpartners are created using one of the techniques discussed above or asknown in the art. Next, chemical libraries and databases are searchedfor molecules similar in structure to the known molecule. That is, asearch can be made of a three dimensional data base for non-peptide(organic) structures (e.g., non-peptide analogs) having threedimensional similarity to the known structure of the target compound.See, e.g., the Cambridge Crystal Structure Data Base, CrystallographicData Center, Lensfield Road, Cambridge, CB2 1EW, England; and Allen, F.H., et al., Acta Crystallogr., B35: 2331-2339 (1979), all of which areexpressly incorporated by reference in their entireties. One programthat allows for such analysis is Insight II having the Ludi module.Further, the Ludi/ACD module allows a user access to over 65,000commercially available drug candidates (MDL's Available ChemicalsDirectory) and provides the ability to screen these compounds forinteractions with HBcAg and/or HBeAg on the computer. The identifiedcandidate binding partners can then be analyzed in a characterizationassay and new molecules can be modeled after the candidate bindingpartners that produce a desirable response. By cycling in this fashion,libraries of molecules that interact HBcAg and/or HBeAg and produce adesirable or optimal response in a characterization assay can beselected.

[0098] It is noted that search algorithms for three dimensional database comparisons are available in the literature. See, e.g., Cooper, etal., J. Comput.-Aided Mol. Design, 3: 253-259 (1989) and referencescited therein; Brent, et al., J. Comput.-Aided Mol. Design, 2: 311-310(1988) and references cited therein, all of which are expresslyincorporated by reference in their entireties. Commercial software forsuch searches is also available from vendors such as Day LightInformation Systems, Inc., Irvine, Calif. 92714, and Molecular DesignLimited, 2132 Faralton Drive, San Leandro, Calif. 94577. The searchingis done in a systematic fashion by simulating or synthesizing analogshaving a substitute moiety at every residue level. Preferably, care istaken that replacement of portions of the backbone does not disturb thetertiary structure and that the side chain substitutions are compatibleto retain the protein:protein interactions.

[0099] Alternatively, these methods can be used to identify improvedbinding partners from an already known binding partner. The compositionof the known binding partner can be modified and the structural effectsof modification can be determined using the experimental and computermodeling methods described above. The altered structure can be comparedto the active site structure of HBcAg and/or HBeAg to determine if animproved fit or interaction results. In this manner systematicvariations in composition, such as by varying side groups, can bequickly evaluated to obtain modified binding partners of improvedspecificity or activity.

[0100] Additionally, a computer model of HBcAg and/or HBeAg can beobtained using the approaches described above, and this model can becompared with libraries of candidate binding partners in real time. Forexample, a search program can locate several structures within thedatabase that have a given set of molecular properties, which correspondto the constraints provided by the HBcAg and/or HBeAg model. With theaid of computer graphics and a retrieval program, candidate bindingpartners can be obtained from the database, modeled, and evaluated forthe ability to interact with HBcAg and/or HBeAg. This approach isreferred to as a “computer generated binding assay”. Such assays can beperformed in the presence or absence of competing molecules.

[0101] A number of articles review computer modelling of drugsinteractive with specific-proteins, such as Rotivinen, et al., 1988,Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist 54-57 (Jun.16, 1988); McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol.29:111-122; Perry and Davies, OSAR: Quantitative Structure-ActivityRelationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989);Lewis and Dean, 1989 Proc. R. Soc. Lond. 236:125-140 and 141-162; and,with respect to a model receptor for nucleic acid components, Askew, etal., 1989, J. Am. Chem. Soc. 111:1082-1090, all of which are expresslyincorporated by reference in their entireties. Other computer programsthat screen and graphically depict chemicals are available fromcompanies such as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc.(Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge,Ontario).

[0102] Many more computer programs and databases can be used withembodiments to identify candidate binding partners and binding partnersthat inhibit viral infectivity and/or modulate a host immune systemresponse. The following list is intended not to limit the invention butto provide guidance to programs and databases that are useful with theapproaches discussed above. The programs and databases that can be usedinclude, but are not limited to: MacPattern (EMBL), DiscoveryBase(Molecular Applications Group), GeneMine (Molecular Applications Group),Look (Molecular Applications Group), MacLook (Molecular ApplicationsGroup), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J.Mol. Biol. 215: 403 (1990), herein incorporated by reference), FASTA(Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988), hereinincorporated by reference), Catalyst (Molecular Simulations Inc.),Catalyst/SHAPE (Molecular Simulations Inc.), Cerius².DBAccess (MolecularSimulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II,(Molecular Simulations Inc.), Discover (Molecular Simulations Inc.),CHARMM (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), Modeller 4 (Sali and Blundell J. Mol. Biol.234:217-241 (1997)), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer (Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), Biopendium (Inpharmatica), SBdBase (Structural Bioinformatics),the EMBL/Swissprotein database, the MDL Available Chemicals Directorydatabase, the MDL Drug Data Report data base, the ComprehensiveMedicinal Chemistry database, Derwents's World Drug Index database, andthe BioByteMasterFile database. Many other programs and data bases wouldbe apparent to one of skill in the art given the present disclosure.

[0103] Although the peptides described above can effectively modulate animmune response to HBV in a subject, binding partner-fusion proteins canbe created to take advantage of already existing highly potent immuneresponses in a subject. That is, peptides that bind HBcAg and/or HBeAgcan be fused with molecules, which are known to elicit a potent immuneresponse in a subject. In this manner, high titer antibodies present inthe subject are redirected to HBcAg and/or HBeAg and, thus, HBV can moreeffectively be cleared from the subject. These binding partner-fusionproteins are referred to as “specificity exchangers” and the sectionbelow describes their manufacture and use in detail.

[0104] Binding Partner Specificity Exchangers

[0105] Antibodies can be redirected to new antigens using bi-functionalsynthetic peptides called binding partner-fusion proteins or specificityexchangers. (See Sällberg et al., Biochemical & Biophysical ResearchCommunications, 205:1386-90 (1994) and U.S. Pat. No. 5,869,232, bothdisclosures are herein incorporated by reference in their entireties).One portion of the specificity exchanger, referred to as the“specificity domain”, comprises a molecule that binds a desired molecule(e.g., a peptide that binds HBcAg and/or HbeAg, such as provided in SEQ.ID. Nos. 5, 16, 17, 28, 29, and 44), which may resemble an antibodybinding domain or fragment thereof and another portion of thespecificity exchanger, called the “antigenic domain”, which serves as anantigen for antibody recognition (preferably recognition by a high titerantibody). The specificity domain can be a binding partner itself (e.g.,peptide, peptidomimetic, or chemical that binds to HbcAg and/or HBeAg),whereas, the antigenic domain can comprise molecules including, but notlimited to, carbohydrates, lipids, proteins, and nucleic acids that haveepitopes, which are recognized by antibodies present in an animal.

[0106] Desirably, the antigenic domain comprises an epitope found on apathogen (e.g., bacteria, mold, fungus, or virus) or a toxin (e.g.,pertussis toxin or cholera toxin) or a non-self antigen. Many antigenicdomains are between 3 and 40 amino acids in length, desirably between 5and 35 amino acids in length, more desirably between 5 and 30 aminoacids in length, preferably between 5 and 25 amino acids in length, andmost preferably between 5 and 20 amino acids in length. The diversity ofantigenic domains that can be used in the specificity exchangers isquite large because a pathogen or toxin can present many differentepitopes. Antigenic domains that comprise an epitope of a pathogen thatalready exists in a subject by virtue of naturally acquired immunity orvaccination are highly desirable. Epitopes of pathogens that causechildhood diseases, for example, can be used as antigenic domains.Epitopes of a pathogen that can be included in an antigenic domain of aspecificity exchanger can include, for example, epitopes on peptidesequences disclosed in Swedish Pat No. 9901601-6; U.S. Pat. No.5,869,232; Mol. Immunol. 28: 719-726 (1991); and J. Med. Virol.33:248-252 (1991); all references are herein expressly incorporated byreference in their entireties.

[0107] Some embodiments have antigenic domains that interact with anantibody that has been administered to the subject. For example, anantibody that interacts with an antigenic domain on a specificityexchanger can be co-administered with the specificity exchanger.Further, an antibody that interacts with a specificity exchanger may notnormally exist in a subject but the subject has acquired the antibody byintroduction of a biologic material (e.g., serum, blood, or tissue). Forexample, subjects that undergo blood transfusion acquire numerousantibodies, some of which can interact with an antigenic domain of aspecificity exchanger. Preferred antigenic domains for use in aspecificity exchanger embodiment comprise viral epitopes including, butnot limited to, the herpes simplex virus, hepatitis B virus, TT virus,and the poliovirus. Highly preferred antigenic domains are provided inTable 6 and the sequence listing (SEQ. ID. Nos. 79-95). TABLE 6Antigenic Domains GLYSSIWLSPGRSYFET (SEQ. ID. No. 79) YTDIKYNPFTDRGEGNM(SEQ. ID. No. 80) DQNIHMNARLLIRSPFT (SEQ. ID. No. 81) LIRSPFTDPQLLVHTDP(SEQ. ID. No. 82) QKESLLFPPVKLLRRVP (SEQ. ID. No. 83) PALTAVETGAT (SEQ.ID. No. 84) STLVPETT (SEQ. ID. No. 85) TPPAYRPPNAPIL (SEQ. ID. No. 86)EIPALTAVE (SEQ. ID. No. 87) LEDPASRDLV (SEQ. ID. No. 88) HRGGPEEF (SEQ.ID. No. 89) HRGGPEE (SEQ. ID. No. 90) VLICGENTVSRNYATHS (SEQ. ID. No.91) KINTMPPFLDTELTAPS (SEQ. ID. No. 92) PDEKSQREILLNKIASY (SEQ. ID. No.93) TATTTTYAYPGTNRPPV (SEQ. ID. No. 94) STPLPETT (SEQ. ID. No. 95)

[0108] In some embodiments, the specificity and antigenic domains aremade separately and are subsequently joined together (e.g., throughlinkers or by association with a common carrier molecule) and in otherembodiments, the specificity domain and antigenic domain are made aspart of the same molecule. For example, any of the binding partners,peptides or specificity domains disclosed herein can be joined to any ofthe antigenic domains of TABLE 6 (SEQ. ID. Nos: 79-95). Although thespecificity and antigenic domains could be made separately and joinedtogether through a linker or carrier molecule (e.g., a complexcomprising a biotinylated specificity domain, streptavidin, and abiotinylated antigenic domain), it is preferred that the specificityexchanger is made as a fusion protein. Thus, preferred embodimentsinclude fusion proteins comprising any of the peptides, bindingpartners, or specificity domains described herein joined to any of theantigenic domains of TABLE 6 (SEQ. ID. Nos:79-95). Thus, preferredspecificity exchangers include, but are not limited to, peptidescomprising, consisting, or consisting essentially of a sequence selectedfrom the group consisting of SEQ. ID. Nos. 5, 6, 13, 16, 17, 28, 29, 33,36, and 37.

[0109] Any of the approaches used to identify and characterize bindingpartners described herein can be used to identify and characterize thespecificity domains of a specificity exchanger specific for HBV. Thespecificity exchangers themselves can also can be analyzed in thecharacterization assays described above or in modified characterizationassays, as will be apparent to one of skill in the art provided thedescription herein. The example below describes the manufacture ofspecificity exchangers having a specificity domain directed to HBcAgand/or HBeAg and an antigenic domain directed to an anti-HSV mAb.

EXAMPLE 7

[0110] An approach to manufacture specificity exchangers that canredirect high titer antibodies to HBV is provided in this example. Afirst set of specificity exchangers having a specificity domaincontaining the HBcAg binding sequence KLSCKASGYIFTS (SEQ. ID. No. 45)and a C terminal antigenic domain containing the epitope for amonoclonal antibody directed to the herpes simplex virus type 1 gG2 (HSVgG2) protein was created. A second set of specificity exchangers havinga specificity domain containing the HBcAg binding sequenceVKLSCKASGYIFTS (SEQ. ID. No. 44) and a C-terminal antigenic domaincontaining the epitope for a monoclonal antibody directed to the HSV gG2protein was also constructed. The sequences of these binding partnerfusion proteins are provided in the Sequence Listing (SEQ. ID. Nos.70-76). These molecules were made by conventional peptide synthesis.

[0111] Once candidate binding partners have been identified, desirably,they are analyzed in a characterization assay. Further cycles ofmodeling and characterization assays can be employed to more narrowlydefine the parameters needed in a binding partner. Each binding partnerand its response in a characterization assay can be recorded on acomputer readable media and a database or library of binding partnersand respective responses in a characterization assay can be generated.These databases or libraries can be used by researchers to identifyimportant differences between active and inactive molecules so thatcompound libraries are enriched for binding partners that have favorablecharacteristics. The section below describes several binding partnercharacterization assays that can be used to evaluate candidate bindingpartners.

[0112] Binding Partner and Candidate Binding Partner CharacterizationAssays

[0113] The evaluation of candidate binding partners and, thus, thedetermination whether a candidate binding partner is, in fact, a bindingpartner can be accomplished by using a “characterization assay”. Theterm “characterization assay” refers to an assay, experiment, oranalysis made on a candidate binding partner or binding partner, whichevaluates the ability of said candidate binding partner or bindingpartner to interact with HBcAg and/or HBeAg or fragments thereof, effectviral infection, and a host immune system response. Encompassed by theterm “characterization assay” are binding studies (e.g., enzymeimmunoassays (EIA), enzyme-linked immunoassays (ELISA), competitivebinding assays, computer generated binding assays, support bound bindingstudies, and one and two hybrid systems), infectivity studies (e.g.,reduction of viral infection, propagation, attachment to a host cell),and analysis of host immune system response e.g., (clearance of viralparticles, reduction in viral lode, activation of antigen presentingcells, and effect on B and T cell presentation). In general, thecharacterization assays can be described in three general catagories:(1) assays that determine whether a candidate binding partner binds toHBcAg and/or HBeAg; (2) assays that determine whether a binding partnerreduces viral infectivity; and (3) assays that determine whether abinding partner modulates a host immune system response.

[0114] Preferred HBcAg and/or HBeAg binding assays use multimericagents. One form of multimeric agent concerns a manufacture comprising acandidate binding partner or binding partner, or fragments thereofdisposed on a support. Another form of multimeric agent involves amanufacture comprising HBcAg and or HBeAg or fragments thereof disposedon a support. These multimeric agents provide the attached molecule insuch a form or in such a way that a sufficient affinity is achieved. A“support” can be a termed a carrier, a protein, a resin, a cellmembrane, or any macromolecular structure used to join or immobilizesuch molecules. Solid supports include, but are not limited to, thewalls of wells of a reaction tray, test tubes, polystyrene beads,magnetic beads, nitrocellulose strips, membranes, microparticles such aslatex particles, animal cells, Duracyte®, artificial cells, and others.A candidate binding partner or binding partner can also be joined toinorganic supports, such as silicon oxide material (e.g. silica gel,zeolite, diatomaceous earth or aminated glass) by, for example, acovalent linkage through a hydroxy, carboxy, or amino group and areactive group on the support.

[0115] In some multimeric agents, the macromolecular support has ahydrophobic surface that interacts with a portion of the candidatebinding partner, binding partner, or viral antigen (e.g., HBcAg and/orHBeAg) by a hydrophobic non-covalent interaction. In some cases, thehydrophobic surface of the support is a polymer such as plastic or anyother polymer in which hydrophobic groups have been linked such aspolystyrene, polyethylene or polyvinyl. Additionally, candidate bindingpartner, binding partner, or viral antigen can be covalently bound tosupports including proteins and oligo/polysaccarides (e.g. cellulose,starch, glycogen, chitosane or aminated sepharose). In these latermultimeric agents, a reactive group on the molecule, such as a hydroxyor an amino group, is used to join to a reactive group on the carrier soas to create the covalent bond. Additional multimeric agents comprise asupport that has other reactive groups that are chemically activated soas to attach the candidate binding partner or binding partner orfragments thereof. For example, cyanogen bromide activated matrices,epoxy activated matrices, thio and thiopropyl gels, nitrophenylchloroformate and N-hydroxy succinimide chlorformate linkages, oroxirane acrylic supports can be used. (Sigma).

[0116] Furthermore, in some embodiments, a liposome or lipid bilayer(natural or synthetic) is contemplated as a support and a candidatebinding partner, binding partner, or viral antigen can be attached tothe membrane surface or are incorporated into the membrane by techniquesin liposome engineering. By one approach, liposome multimeric supportscomprise a candidate binding partner, binding partner, or viral antigenthat is exposed on the surface. A hydrophobic domain can be joined tothe candidate binding partner or binding partner so as to facilitate theinteraction with the membrane.

[0117] Supports for use in the body, (i.e. for prophylactic ortherapeutic applications) are desirably physiological, non-toxic andpreferably, non-immunoresponsive. Suitable carriers for use in the bodyinclude poly-L-lysine, poly-D, L-alanine, liposomes, and Chromosorb®(Johns-Manville Products, Denver Colo.). Ligand conjugated Chromosorb®(Synsorb-Pk) has been tested in humans for the prevention ofhemolytic-uremic syndrome and was reported as not presenting adversereactions. (Armstrong et al. J. Infectious Diseases 171:1042-1045(1995)). For some embodiments, a “naked” carrier (i.e., lacking anattached binding partner) that has the capacity to attach a bindingpartner in the body of a organism is administered. By this approach, a“prodrug-type” therapy is envisioned in which the naked carrier isadministered separately from the binding partner and, once both are inthe body of the organism, the carrier and the binding partner areassembled into a multimeric complex.

[0118] The insertion of linkers, such as linkers (e.g., “λ linkers”engineered to resemble the flexible regions of λ phage) of anappropriate length between the candidate binding partner, bindingpartner, or viral antigen and the support are also contemplated so as toencourage greater flexibility of the candidate binding partner, bindingpartner, or viral antigen and thereby overcome any steric hindrance thatcan be presented by the support. The determination of an appropriatelength of linker that allows for optimal binding to HBcAg and/or HBeAg,inhibition of viral infectivity, and modulation of host immune responsecan be determined by screening the attached molecule with varyinglinkers in the characterization assays detailed in the presentdisclosure.

[0119] A composite support comprising more than one type of bindingpartner is also envisioned. A “composite support” can be a carrier, aresin, or any macromolecular structure used to attach or immobilize twoor more different binding partners. In some embodiments, a liposome orlipid bilayer (natural or synthetic) is contemplated for use inconstructing a composite support or binding partners are attached to themembrane surface or are incorporated into the membrane using techniquesin liposome engineering.

[0120] As above, the insertion of linkers, such as λ linkers, of anappropriate length between the binding partner and the support is alsocontemplated so as to encourage greater flexibility in the molecule andthereby overcome any steric hindrance that can occur. The determinationof an appropriate length of linker that allows for optimal binding toHBcAg and/or HBeAg, inhibition of viral infectivity, and modulation ofhost immune response or lack thereof, can be determined by screening thebinding partners with varying linkers in the characterization assaysdetailed in the present disclosure.

[0121] Several approaches to identify agents that interact with HBcAgand/or HBeAg, employ a multimeric support having candidate bindingpartner, binding partner, or HBcAg and/or HBeAg or a fragment thereof.For example, support-bound candidate binding partner can be contactedwith “free” HBcAg and/or HBeAg and an association can be determineddirectly (e.g., by using labeled HBcAg and/or HBeAg) or indirectly(e.g., by using a labeled antibody directed to the HBcAg and/or HBeAg).Thus, candidate binding partners are identified as binding partners byvirtue of the association of HBcAg and/or HBeAg with the support-boundcandidate binding partner. Alternatively, support-bound HBcAg and/orHBeAg can be contacted with “free” candidate binding partner and theamount of associated candidate binding partner can be determineddirectly (e.g., by using labeled binding partner) or indirectly (e.g.,by using a labeled antibody directed to the binding partner).

[0122] Assays that determine whether a candidate binding partner bindsto HBcAg and/or HBeAg can be conducted in a variety of formats. Mostsimply, techniques in immunology can be employed or readily adapted toascertain whether a candidate binding partner has bound HBcAg and/orHBeAg. In one set of characterization assays, the interaction of 20amino acid long candidate binding partners with HBcAg was determined byenzyme immunoassays (EIA), as described in the example below.

EXAMPLE 8

[0123] To determine whether a candidate binding partner bound to HBcAgand/or HbeAg, a multimeric support-based binding assay was performed.Accordingly, synthetic peptides corresponding to the VH and VL domainsof the sequenced mAbs were passively adsorbed to 96 well microplates inserial dilutions starting at 200 μg/ml. The peptide coated plates werethen incubated with HBcAg and HBeAg serially diluted in PBS-GT. Theamount of HBcAg and HBeAg bound by the peptides was then detected usinga mAb directed to an epitope common to HBcAg and HBeAg (See Sällberg etal., J General Virology 74:1335-1340 (1993), herein expresslyincorportaed by reference in its entirety) diluted 1:3000 in PBS-GT. Thequantity of bound mAb was determined by addition by peroxidase labeledrabbit anti-mouse IgG (P260, Dako AS, Denmark). The plates weredeveloped by incubation with ortho-phenylene-diamine (Sigma) and theabsorbance at 492 nm was evaluated.

[0124] As shown in TABLE 7, many peptides were found to bind to HBcAg inthis characterization assay. Two conserved domains were found in five ofthe binding partners (SEQ. ID. Nos. 5, 16, 17, 28, and 29), which boundHBcAg with high affinity. These conserved domains were Cys-Lys-Ala-Ser(SEQ. ID. No. 77) and Cys-Arg-Ala-Ser (SEQ. ID. No. 78). Taken together,the levels of affinity of the five binding partners and the conservationof the two domains provided evidence that these domains are intimatelyinvolved in binding to HBcAg. TABLE 7 Binding of support-bound peptidesto HBcAg and NS3 OD at 405 nm to indicated antigen HBcAg HBcAg HCVPeptide # Sequence of peptide 2 μg/well 0.2 μg/well NS3 MAb 4-2 1VKLQQSGTEVVKPGASVKLS (SEQ. ID. No. 4) 0.323 0.124 0.021 2VKPGASVKLSCKASGYIFTS (SEQ. ID. No. 5) 3.692 1.146 0.269 3CKASGYIFTSYDIDWVRQTP (SEQ. ID. No. 6) 0.525 0.187 0.032 4YDIDWVRQTPEQGLEWIGWI (SEQ. ID. No. 7) 0.551 0.202 0.089 5EQGLEWIGWIFPGEGSTEYN (SEQ. ID. No. 8) 0.706 0.256 0.182 6FPGEGSTEYNEKFKGRATLS (SEQ. ID. No. 9) 0.325 0.121 0.109 7EKFKGRATLSVDKSSSTAYM (SEQ. ID. No. 10) 0.883 0.194 0.035 8VDKSSSTAYMELTRLTSEDS (SEQ. ID. No. 11) 0.363 0.134 0.041 9ELTRLTSEDSAVYFCARGDY (SEQ. ID. No. 12) 0.574 0.195 0.073 10AVYFCARGDYDYYRRYFDLW (SEQ. ID. No. 13) 0.981 0.304 0.038 11DYYRRYFDLWGQGTTVTVS (SEQ. ID. No. 14) 0.356 0.133 0.022 MAb 5H7 12DIVLTQSPASLAVSLGQRAT (SEQ. ID. No. 15) 0.53 0.156 0.025 13LAVSLGQRATISCRASQSVS (SEQ. ID. No. 16) 3.113 0.807 0.128 14ISCRASQSVSTSSYSYMHWY (SEQ. ID. No. 17) 2.475 0.449 0.156 15TSSYSYMHWYQQKPGQPPKL (SEQ. ID. No. 18) 1.442 0.574 0.028 16QQKPGQPPKLLIKYASNLES (SEQ. ID. No. 19) 0.299 0.064 0.016 17LIKYASNLESGVPARFSGSG (SEQ. ID. No. 20) 0.357 0.112 0.020 18GVPARFSGSGSGTDFTLNIH (SEQ. ID. No. 21) 0.409 0.141 0.027 19SGTDFTLNIHPVEEEDTATY (SEQ. ID. No. 22) 0.649 0.205 0.206 20PVEEEDTATYYCQHSWEIPY (SEQ. ID. No. 23) 0.625 0.207 0.124 21YCQHSWEIPYTFGGGTKLEI (SEQ. ID. No. 24) 0.498 0.173 0.052 22TFGGGTKLEIKRADAAPAV (SEQ. ID. No. 25) 0.273 0.084 0.024 23KRADAAPAVSIFPPSSKLG (SEQ. ID. No. 26) 0.465 0.162 0.021 MAb 9C8 24IQLQQSGAELVKPGASVKIS (SEQ. ID. No. 27) 0.369 0.123 0.021 25VKPGASVKISCKASGYSFTG (SEQ. ID. No. 28) 3.129 0.845 0.097 26CKASGYSFTGYNMNWVKQSH (SEQ. ID. No. 29) 2.29 0.356 0.053 27YNMNWVKQSHGKSLEWIGNI (SEQ. ID. No. 30) 0.157 0.114 0.021 28GKSLEWIGNINPYYGSTSYN (SEQ. ID. No. 31) 0.289 0.114 0.028 29NPYYGSTSYNQKFKGKATLT (SEQ. ID. No. 32) 0.783 0.213 0.021 30QKFKGKATLTVDKSSSTAYM (SEQ. ID. No. 33) 1.115 0.207 0.035 31VDKSSSTAYMQLNSLTSEDS (SEQ. ID. No. 34) 0.338 0.114 0.106 32QLNSLTSEDSAVYYCARGKG (SEQ. ID. No. 35) 0.528 0.121 0.035 33AVYYCARGKGTGFAYWGQGT (SEQ. ID. No. 36) 1.203 0.227 0.035 34TGFAYWGQGTLVTVSAAKTT (SEQ. ID. No. 37) 0.898 0.192 0.035 35LVTVSAAKTTPPSVYPLVPV (SEQ. ID. No. 38) 0.59 0.198 0.032

[0125] The five “high affinity” binding partners (SEQ. ID. Nos. 5, 16,17, 28, and 29) were also bound to a support and were analyzed for theability to bind various dilutions of “free” HBcAg and HBeAg. As shown inTABLE 8, the five peptides exhibited appreciable binding to HBcAg andHBeAg at concentrations as low as 0.67 μg/ml. The converse of an aspectof this experiment was also performed. That is, various dilutions ofsupport-bound binding partners (SEQ. ID. Nos. 5, 16, 17, 28, and 29)were contacted with 10 μg/ml of HBcAg and binding was evaluated. Asshown in TABLE 9, the peptides of SEQ. ID. Nos. 5, 16, 17, and 28 showedappreciable binding at low concentrations of HBcAg and/or HBeAg. In eachof these experiments, the amount of peptide binding was directlyproportional to the amount of HBcAg or HBeAg added or the amount ofpeptide disposed on the support. TABLE 8 Binding of dilutions of HBcAgand HBeAg to support-bound peptides Peptide Amount HBcAg added (μg/ml)Amount HBeAg added (μg/ml) # 20 10 5 2.5 1.25 0.67 20 10 5 2.5 1.25 0.67 2 3.453 1.692 0.989 0.374 0.155 0.034 0.751 0.222 0.096 0.063 0.0430.081 13 2.635 0.872 0.501 0.195 0.079 0.028 0.393 0.060 0.038 0.0300.023 0.024 14 2.660 1.150 0.633 0.252 0.117 0.039 0.687 0.246 0.1320.102 0.056 0.046 25 2.652 0.897 0.359 0.159 0.058 0.036 0.319 0.0960.050 0.115 0.034 0.022 26 1.479 0.601 0.245 0.101 0.038 0.028 0.2410.091 0.091 0.063 0.026 0.027 27 0.305 0.115 0.059 0.042 0.026 0.0200.040 0.037 0.029 0.032 0.021 0.018

[0126] TABLE 9 Binding of 10 μg/ml HBcAg to dilutions of support-boundpeptides Peptide Amount peptide coated (μg/ml) # 100 50 25 12.5 6.253.125 2 1.453 1.218 1.039 0.913 0.597 0.333 13 0.900 0.434 0.276 0.2400.388 0.355 14 0.641 0.569 0.636 0.514 0.630 0.413 25 1.011 0.766 0.5690.423 0.298 0.217 26 0.422 0.198 0.181 0.179 0.156 0.171 27 0.151 0.1700.162 0.181 0.175 0.168

[0127] Variations of the characterization assays described above includecompetitive binding assays. For example, the five high affinity peptides(SEQ. ID. Nos. 5, 16, 17, 28, and 29) were analyzed for the ability toprevent binding of mAb 4-2 to HBcAg. Initial experiments were conductedby contacting binding partner to HBcAg prior to introducing theantibody. As shown in TABLE 10, all five high affinity binding partnerswere able to inhibit binding of mAb 4-2 when the concentration ofpeptide was raised to 200μg/ml. The peptides of SEQ. ID. Nos. 5, 28, and29 effectively reduced binding of the antibody at concentrations as lowas 100 μg/ml and the peptides of SEQ. ID. No. 5 and 29 appreciablyreduced binding of mAb 4-2 at concentrations as low as 50 μg/ml. Theability of the five high affinity binding partners to compete forbinding of the mAb 4-2 to HBcAg was also analyzed. When the peptides ofSEQ. ID. Nos. 5, 16, 28, and 29 were provided at 200 μg/ml in thepresence of mAb 4-2, the binding of the antibody to HBcAg wassignificantly inhibited. (See TABLE 11). Similarly, the peptides of SEQ.ID. Nos. 5, 16, 28, and 29 effectively competed with mAb 4-2 at 100μg/ml. The peptide of SEQ. ID. No. 28 prevented binding of mAb 4-2 at aconcentration as low as 12.5 μg/ml, whereas, the peptides of SEQ. ID.Nos. 5 and 16 prevented binding of mAb 4-2 at 6.25 μg/ml. TABLE 10Inhibition of mAb 4-2 binding to HBcAg coated at 1 μg/ml by priorincubation with synthetic peptides*. Peptide Amount peptide (μg/ml)added prior to mAb 4-2 # 200 100 50 25 12.5 6.25 2 0.078 0.083 0.1880.341 0.331 0.336 13 0.299 0.345 0.364 0.429 0.365 0.350 14 0.305 0.3700.402 0.439 0.380 0.370 25 0.173 0.274 0.312 0.359 0.339 0.329 26 0.2010.280 0.337 0.383 0.357 0.351 16 0.368 0.394 0.383 0.406 0.391 0.336

[0128] TABLE 11 Inhibition of mAb 4-2 binding to HBcAg coated at 5 μg/mlby simultaneous addition of mAb and synthetic peptides*. Peptide Amountpeptide (μg/ml) added prior to mAb 4-2 # 200 100 50 25 12.5 6.25 2 0.1390.194 0.395 0.698 0.824 0.867 13 0.859 0.891 0.998 1.027 1.037 0.979 141.468 1.458 1.557 1.390 1.442 1.264 25 0.302 0.910 1.217 1.200 1.2071.178 26 0.341 0.947 1.250 1.347 1.275 1.196 16 0.925 1.115 1.232 1.2391.235 1.065

[0129] Preferably, the specificity exchangers are also evaluated incharacterization assays. Some of these characterization assays evaluatethe ability of the specificity exchanger to interact with the targetmolecule and the redirecting antibody. Other characterization assaysevaluate the ability of the specificity exchanger to fix complement.Still more characterization assays are designed to determine whether aspecificity exchanger can bind to the target molecule, bind to theredirecting antibody, and fix complement. The example below describesseveral characterization assays that were conducted on the specificityexchangers.

EXAMPLE 9

[0130] To evaluate the efficacy of the specificity exchangers describedabove, several binding assays using a mAb specific for HSV gG2 wereconducted. In one assay, the various specificity exchangers were coatedonto microtiterplates and were bound with the mAb specific for HSV gG2.As shown in TABLE 12, the specificity exchangers provided in SEQ. ID.Nos. 71, 74, and 75 appreciably bound the mAb. In another assay, HBcAgwas coated onto microtiter plates, specificity exchanger was added (20μg/well), and the binding of the mAb specific for HSV gG2 wasdetermined. The specificity exchangers provided in SEQ. ID. Nos. 71 and74 appreciably bound the immobilized HBcAg and allowed for the bindingof the mAb specific for HSV gG2. In another binding assay, HBeAg wascoated onto microtiter plates, specificity exchanger was added (20μg/well), and the binding of the mAb specific for HSV gG2 wasdetermined. Similarly, the specificity exchangers provided in SEQ. ID.Nos. 71 and 74 appreciably bound the immobilized HBeAg and allowed forthe binding of the mAb specific for HSV gG2. These results demonstratethat specificity exchangers specific for HBcAg and/or HBeAg can bemanufactured and used to redirect HBV to high titer antibodies. Thecharacterization assays used to evaluate binding partners can also beused and/or modified for the analysis of the specificity exchangers.TABLE 12 Redirection of antibodies specific for an epitope within HSVgG2 gG2 mAb binding to indicated antigen on solid phase fusion peptideHBcAg HBeAg Addition of fusion peptide in solution (20 μg/well) Peptide# Fusion peptide sequence no yes yes 13 KLSCKASGYIFTSEHRGGPEE (SEQ. ID.No. 70) 0.040 0.020 0.022 14 KLSCKASGYIFTSHRGGPEEF (SEQ. ID. No. 71)1.622 0.550 0.638 15 KLSCKASGYIFTSRGGPEEFE (SEQ. ID. No. 72) 0.062 0.0160.040 16 VKLSCKASGYIFTSEHRGGPE (SEQ. ID. No. 73) 0.060 0.038 0.029 17VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74) 0.884 1.462 1.727 18VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 75) 0.213 0.037 0.032 19VKLSCKASGYIFTSGGPEEFE (SEQ. ID. No. 76) 0.018 0.025 0.024 Neg ctrlPeptide 16 Not tested 0.038 0.022

[0131] In another experiment, it was found that binding of the peptideVKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74) to HBcAg can be inhibited by apeptide derived from HBcAg. That is, the precise domain on HbcAg thatbinds to the peptide VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74) wasdiscovered. In this experiment, microplates were coated overnight with 1μg/ml HBcAg and, subsequently, a 100 μl mixture containing 5 μg of thepeptide VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74) and 5 μg of an HBcAgpeptide was added to the coated plate. The HBcAg peptides wereapproximately 20 amino acids in length and corresponded to regions ofHBcAg spanning amino acid residues 11-183. The experiment was performedin triplicate. The amount of bound peptide (SEQ. ID. No. 74) was thendetermined by adding a 1:1000 dilution of an HSV gG2 mAb followed by theaddition of enzyme conjugated anti-mouse Ig, which interacts with theHSV gG2 mAb. The amount of enzyme present, which directly correlates tothe amount of peptide (SEQ. ID. No. 74) that bound to the coated plates,was then determined by adding the substrate ortho-phenylene diamine andanalyzing the absorbance at 490 nm. As shown in TABLE 13, the HBcAgpeptide SRDLVVSYVNTNMGLKFRQL (SEQ. ID. NO. 103) and TNMGLKFRQLLWFHISCLTF(SEQ. ID. No.104), which correspond to amino acid residues 81-100 and91-110 of HbcAg, respectively consistently reduced the binding of thepeptide VKLSCKASGYIFTSHRGGPEE (SEQ. ID. No. 74). This result indicatedthat the HBcAg-binding domain of the peptide VKLSCKASGYIFTSHRGGPEE (SEQ.ID. No. 74) involves amino acid residues 91-1 10 of HBcAg.

[0132] Thus, embodiments also include complexes of binding partners withHBcAg, HBeAg, HBV capsids, and HBV itself or fragments thereof.Preferred embodiments include complexes of binding partner and peptidescomprising, consiting essentially of or consisting of either SEQ. ID.No. 103 or SEQ. ID. No. 104. Highly preferred embodiments includecomplexes comprising a peptide comprising, consiting essentially of orconsisting of the peptide of SEQ. ID. No. 74 joined to either SEQ. ID.Nos. 103 or 104. Additional embodiments include peptides comprising,consiting essentially of or consisting of the sequence of SEQ. ID. Nos.103 or 104 or nucleic acids encoding these molecules. These embodimentsare valuable research tools and can be used as active ingredients invaccines or therapeutics. TABLE 13 Std. Corresponding Deviation residueswithin OD at from the Sequence of inhibiting HBcAg peptide HBcAgSequence ID 490 nm mean none — — 1.454 0.831 ATVELLSFLPSDFFPSVRDL 11-3096 1.387 0.696 SDFFPSVRDLLDTASALYRE 21-40 97 1.576 1.077LDTASALYREALESPEHCSP 31-50 98 1.589 1.103 ALESPEHCSPHHTALRQAIL 41-60 991.372 0.665 HHTALRQAILCWGELMTLAT 51-70 100 0.646 −0.798CWGELMTLATWVGVNLEDPA 61-80 101 1.395 0.712 WVGVNLEDPASRDLVVSYVN 71-90102 1.411 0.744 SRDLVVSYVNTNMGLKFRQL  81-100 103 0.386 −1.323TNMGLKFRQLLWFHISCLTF  91-110 104 0.058 −1.984 LWFHISCLTFGRETVIEYLV101-120 105 1.129 .175 GRETVIEYLVSFGVWIRTPP 111-131 106 0.832 −0.423SFGVWIRTPPAYRPPNAPIL 121-140 107 0.701 −0.688 AYRPPNAPILSTLPETTVVR131-150 108 1.564 1.052 STLPETTVVRRRGRSPRRRT 141-160 109 0.811 −0.466RRGRSPRRRTPSPRRRRSQS 151-170 110 0.732 −0.625 PSPRRRRSQSPRRRRSQSRESQC161-183 111 0.678 −0.734 Mean OD ± SD — — 1.042 ± 0,469 —

[0133] To demonstrate that the biological activities encompassed by theFc domain of the HSV mAb was introduced to the specificity exchanger(SEQ. ID. No. 74), a complement binding assay was performed.Accordingly, the assay was conducted by mixing the antibody to be testedwith the appropriate antigen and allowing binding to occur overnight.Subsequently, rabbit complement, which is consumed if the antibody bindsthe antigen and the complement, was added. To determine the amount ofresidual complement in the mixture, sheep erythrocytes and an antibodyto sheep erythrocytes was added. If the complement has been exhausted(i.e. a complement binding antibody has bound to the antigen) lysis ofred blood cells will not occur. If no antibody-antigen complex hasformed then the remaining complement will lyse the red blood cells boundby the specific antibody.

[0134] The complement binding of the HBcAg-specific mAb 9C8 wasevaluated together with the ability of the specificity exchanger peptide(SEQ. ID. No. 74) to inhibit the binding of mAb 9C8 to HBcAg. Serialdilutions of mAb 9C8 were mixed with HBcAg in the presence and absenceof the specificity exchanger peptide (SEQ. ID. No. 74). As shown inTABLE 14, the mAb 9C8 bound to HBcAg appreciably bound complement andthe specificity exchanger peptide (SEQ. ID. No. 74) was unable tocompete away bound mAb 9C8. TABLE 14 Complement binding activity of mAb9C8 in the presence of a specificity exchanger Amount peptide AmountSEQ. ID. No. 74 HBcAg Amount mAb 9C8 (pmol) (pmol) (pmol) 6.7 3.3 1.70.8 none none − − − − none 24 + + − − 4000 24 + + + −  400 24 + + − ? 40 24 + + − −   4 24 + + − −   0.4 24 + + − −

[0135] The complement binding of mAb 4-2 was also evaluated inconjunction with the ability of the specificity exchanger peptide (SEQ.ID. No. 74) to inhibit binding of mAb 4-2 to HBcAg. Accordingly, serialdilutions of mAb 4-2 were mixed with HBcAg in the presence and absenceof the specificity exchanger peptide (SEQ. ID. No. 74). As shown inTABLE 15, the mAb 4-2 bound to HBcAg also bound complement. Further, thespecificity exchanger (SEQ. ID. No. 74) appreciably inhibited thebinding of mAb 4-2 to HBcAg. TABLE 15 Complement binding activity of mAb4-2 in the presence of a specificity exchanger Amount peptide AmountSEQ. ID. No. 74 HBcAg Amount 4-2 mAb (pmol) (pmol) (pmol) 6.7 3.3 1.70.8 none none − − − − none 24 +/− + − − 4000 24 − − − −  400 24 − − − − 40 24 − − − −   4 24 +/− − − +/−   0.4 24 +/− +/− − −

[0136] The ability of the complex of HSV mAb, specificity exchanger(SEQ. ID. No. 74), and HBcAg to bind complement was also evaluated.Accordingly, serial dilutions of the HSV mAb were mixed with HBcAg inthe presence and absence of the specificity exchanger (SEQ. ID. No. 74).As shown in TABLE 16, the HSV mAb did not activate complement in theabsence of the specificity exchanger (SEQ. ID. No. 74). However, whenthe HSV mAb and the specificity exchanger (SEQ. ID. No. 74) were presentat equimolar ratios, the HSV mAb bound HBcAg through the specificityexchanger (SEQ. ID. No. 74) and the mAb-specificity exchanger-HBcAgcomplex was able to bind complement These data confirm that an antibodybound to the specificity exchanger (SEQ. ID. No. 74) can impart thebiological activity of the antibody to the specificity exchangerpeptide. TABLE 16 Complement binding activity of a specificity exchangerbound to HBcAg and a HSV mAb Amount peptide Amount Amout HSV mAb (pmol)SEQ. ID. No. 74 HBcAg Excess Excess Excess Excess (pmol) (pmol) 6.7peptide 3.3 peptide 1.7 peptide 0.8 peptide none none − − − − None 24 −− − − 4000 24 +/− 597 − 1212 − 2352 − 5000  400 24 − 59.7 − 121.2 −235.2 + 500  40 24 − 5.97 − 12.12 − 23.52 − 50   4 24 − 0.597 +/−1.212 + 2.352 − 5   0.4 24 − 0.0597 − 0.1212 +/− 0.2352 − 0.5

[0137] In another type of characterization assay, the ability of abinding partner to inhibit viral infectivity is analyzed. Several typesof HBV viral infectivity assays can determine the efficacy of anti-HBVmaterials by detecting a reduction in HBV nucleic acid synthesis and/orthe presence of HBV antigens. These assays can be readily adapted todetermine whether a binding partner inhibits HBV propagation. By oneapproach, a human hepatoblastoma cell culture assay is used to evaluatethe ability of binding partners to inhibit HBV replication. (See Korbaand Gerin, Antiviral Res. 19: 55-70 (1992) and Korba and Milman,Antiviral Res. 15:217-228 (1991), both references herein incorporated byreference in their entirety). By another approach, persistently infectedHepG2 cells can be used to evaluate the ability of binding partners toinhibit HBV replication. The toxicity of binding partners can also beassessed under the same culture and treatment conditions. The examplebelow describes several characterization assays that can be used todetermine the ability of a binding partner to inhibit HBV infectivity.

EXAMPLE 10

[0138] A human hepatoblastoma cell culture assay can be used to evaluatethe ability of a binding partner to inhibit HBV replication. Thedetection of HBV propagation is determined by analyzing the presence ofHBV nucleic acid in the media and cells. Human hepatoblastoma 2.2.15cells are grown to confluency and are provided a daily dose of bindingpartner in RPMI1640 medium with 2% fetal bovine serum. Ideally, atitration of binding partner (e.g., 500 μM to 1 μM) is provided toaliquots of cells. As a control, scrambled peptides having a similaramino acid content are added at the same concentrations. Medium isanalyzed for HBV virion DNA before treatment and daily during treatment.HBV DNA can be extracted from medium and analyzed by slot blot analysis,for example. Intracellular HBV DNA is also analyzed after 10 days oftreatment. Preferably, the cellular DNA is prepared and analyzed bySouthern blot analysis using a ³²P-labelled 3.2 kb EcoRI HBV DNAfragment as a probe. Quantitation can be accomplished by comparison toHBV standards loaded on each gel. Desirable binding partners willinhibit HBV propagation by greater than 50%, as demonstrated by areduction of HBV virion DNA in the medium and 3.2 Kb DNA syntheses.

[0139] Additionally, the effect of a binding partner on HBV propagationin HEP-G2 cells can be monitored by detecting the presence of HBVantigens (e.g., HBsAg and HBeAg) in the media. Kits that detect thesehepatitis markers are commercially available. (Abbott Laboratories). Asabove, human hepatoblastoma 2.2.15 cells are grown to confluency and areprovided a daily dose (200 μg/ml) of binding partner in RPMI1640 mediumwith 2% fetal bovine serum. Medium is assayed for HBV virion DNA beforetreatment and periodically during treatment for the presence of a viralantigen by using a commercially available kit (Abbott Laboratories).Desirable binding partners will inhibit HBV propagation by greater than50%, as demonstrated by a reduction of viral antigen present in themedium.

[0140] By another approach, HEP-G2 cells, persistently infected withHBV, are treated daily with fresh D-MEM containing 20% FBS and atitration of binding partner, as above. After 9 days incubation andtreatment, the cells and overlay medium are harvested separately toassay the quantity of nucleic acids. Extracellular virion DNA inuntreated cells will range from from 50 to 150 pg/ml in the overlaymedium. Intracellular HBV DNA replication intermediates (RI) inuntreated cells will range from 50 to 100 pg/μg cell DNA. Hybridizationanalysis will show that approximately 1 pg intracellular HBV DNA/igcellular DNA to 2-3 genomic copies per cell and 1.0 pg of extracellularHBV DNA/ml overlay medium to 3×10³ viral particles/ml. HBV RNA can alsobe analyzed by Northern blot hybridization analysis using a ³²P-labeled3.2 Kb gel-purified cloned genomic probe. Quantitive analysis ofintracellular HBV DNA and 14BV RNA can be performed using an AMBIS betascanner. Desirable binding partners will inhibit the level of HBVpropagation demonstrated by untreated cells by a value of greater than50%, as demonstrated by a reduction of virion replication intermediates(RI) and 3.2 Kb DNA syntheses.

[0141] Additionally, the effect of a binding partner on HBV propagationin a human hepatoblastoma cell culture assay is monitored by detectingthe presence of HBV antigens (e.g., HBsAg and HBeAg) in the media. Kitsthat detect these hepatitis markers are commercially available. (AbbottLaboratories). HEP-G2 cells are persistently infected with HBV and aretreated daily with fresh D-MEM containing 20% FBS and 200 μg/ml bindingpartner. After 9 days of incubation and treatment, the overlay medium isharvested to assay the quantity of HBsAg and HBeAg by EIA (AbbottLaboratories). The overlay medium is diluted to levels of antigen in thelinear range of the assay. Standard curves using dilutions of positiveHBsAg and HBeAg controls are included in each assay. Desirable bindingpartners will inhibit HBV propagation by greater than 50%, asdemonstrated by a reduction of viral antigen present in the medium.

[0142] Toxicity of a binding partner can also be determined by theexclusion of neutral red dye uptake in cells grown in 96-well plates andtreated as described above. One day after the final addition of bindingpartner, medium is removed and 0.2 ml of DPBS containing 0.01% neutralred dye (Sigma, Inc.) is added to each well. Cells are allowed torecover for two hours. Dye is removed, cells are washed with DPBS andthen 0.2 ml of 50% EtOH/1% glacial acetic acid is added to each well.After 30 minutes of gentle mixing, absorbance at 510 nm is measured andcompared to untreated control cultures. Desirable binding partners willdemonstrate a toxicity of less than 5% at concentrations ten foldgreater than that shown to be effective at inhibiting HBV propagation.The in vitro assays described in this example can be used to rapidlydetermine whether a binding partner can inhibit HBV infection.

[0143] Characterization assays also include experiments designed to testbinding partners in vivo. There are many animal models that are suitablefor evaluating the ability of a binding partner to inhibit HBVinfection. The woodchuck model has been used in hepatitis research forover a decade. (See e.g., Gerin, J. L. 1984. In Advances in HepatitisResearch. F. Chisari, ed. Masson Publishing USA, Inc. New York, pp.40-48; Gerin et al. 1986 In Vaccines 86: New approaches to Immunization.F. Brown et al., eds. Cold Spring Harbor Laboratory Press, N.Y., pg383-386, herein expressly incorporated by reference in its entirety).The woodchuck hepatitis virus (WHV) is closely related to HBV, bothimmunologically and in terms of sequence homology. Woodchucks are nowbred and reared for experimental hepatitis research. Infection of younganimals with defined WHV inocula yields chronic carriers for drugtesting and research. At least one commercial testing facility isdevoted to testing of compounds in woodchucks. Tennant, B. C. and J. L.Gerin. 1994. In The Liver: Biology and Pathobiology, Third Edition. I.M. Arias et al., eds. Raven Press, Ltd., N.Y. pp 1455-1466, hereinexpressly incorporated by reference in its entirety. Because of thesequence homology between HBV and WHV, the efficacy of the bindingpartners can be evaluated in the woodchuck model. Furthermore,demonstration of binding partner efficacy in this model is a cleardemonstration of a specific pharmacologic effect to those of skill inthe art.

[0144] A more recently developed animal model for HBV uses transgenicrats that express human hepatitis B virus genes. (See e.g., Takahashi etal., Proc. Natl. Acad. Sci. U.S.A. 92, 1470-1474 (1995), hereinexpressly incorporated by reference in its entirety). These animalsdevelop acute hepatitis and viral particles and HBeAg are seen in theblood between three and seven days after transfection. HBV is expressedin the liver and liver cell death results. These effects and thesubsequent clearing of virions from the blood mimic the effects of acuteHBV infection in humans. Therefore activity of binding partners in thismodel is indicative of therapeutic activity in humans to those of skillin the art.

[0145] Chimpanzees are hosts for HBV, and therefore constitute anotheranimal model for HBV induced disease. The serological events followinginfection in chimpanzees are identical to that observed in humans. Bothacute and chronic infections result from exposure of chimpanzees to HBV.However, chimpanzees do not have recognizable clinical symptoms ofhepatitis. Cornelius, C. E., 1988, in The Liver: Biology andPathobiology, Second Ed. I. M. Arias et al., eds. Raven Press, Ltd.,N.Y., pp. 1315-1336, herein expressly incorporated by reference in itsentirety. Demonstration of activity in this model, in which the animalis infected with the same virus that infects humans, is also indicativeof therapeutic effect in humans to those skilled in the art. The examplebelow describes in vivo assays that can be performed in woodchucks todetermine whether a binding partner can inhibit HBV infection.

EXAMPLE 11

[0146] Binding partners can be evaluated for their ability to inhibitHBV infection at a commercial facility, which routinely screens anti-HBVand anti-hepatocellular carcinoma drug candidates in the woodchuckhepatitis model. (Marmotech, Inc. of Ithaca, N.Y.). Two doses of bindingpartner are tested, 20 mg/kg and 2 mg/kg, with three animals receivingeach dose. Binding partners are administered intravenously in 0.1 ml ofPBS every other day for 30 days, for a total of 15 doses. The primaryend point of the assay is level of circulating virus. Blood samples arecollected on day 0, prior to drug treatment, and at days 1, 2, 4, 8, 15,22 and 30 of treatment. Virus is quantitated by dot blot or Southernblot analysis using the methods described above or by monitoring thepresence of viral antigens in the blood using a commercially availablekit (Abbott Laboratories). Alternatively, binding partners can beevaluated in rats or mice made transgenic for HBV genes. (See Takahashiet al., Proc. Natl. Acad. Sci. U.S.A., 92:1470-1474 (1995), hereinincorporated by reference in its entirety). The next example describesan approach that was used to evaluate the efficacy of a binding partner(e.g., a specificity exchanger) for use as the active ingredient in apharmaceutical that is administered to treat or prevent HBV infection.

EXAMPLE 12

[0147] By using an animal model of HBV infection, the therapeuticefficacy of a specificity exchanger directed to HBV was determined. In afirst group of experiments, two HBeAg-transgenic mice (obtained from Dr.David R Milich, The Scripps Research Institute, La Jolla, CA) wereinjected ip. with 500 μl of undiluted hybridoma supernatants. The micewere then bled on day zero, two, six and nine post-injection. Sera werethen tested for the change in serum HBeAg levels as determined by EIA(Sorin Biomedica, Saluggia, Italy). As shown in TABLE 17, neither PBS ormAb 3-4 had an effect on the HBeAg levels in serum. In contrast, mAb 4-2complexed an appreciable amount of serum HBeAg (i.e., over a two foldincrease in serum HBeAg was detected by EIA). This data confirms thatmAb 4-2 binds HBeAg in vivo at an epitope that does not compete with theantibodies present in the commercial EIA. TABLE 17 In vivo aggregationof serum HBeAg in transgenic mice by mAbs 3-4 and 4-2* Fold change inserum HBeAg levels as determined by EJA in HBeAg-Tg Mab given mice atindicated day from injection in vivo 0 2 6 9 PBS 1 0.8 1.1 1 3-4 1 0.81.1 1.1 4-2 1 2.7 2.2 1.7

[0148] In a second group of experiments, two HBeAg-transgenic mice wereinjected ip. with either culture media (RPMI), the HSV mAb, theHBcAg-specific mab 9C8, or the specificity exchanger peptide (SEQ. ID.No. 74). As shown in TABLE 18, the negative controls RPMI and the HSVmAb alone show only a 1.2 to 1.9 fold changes in aggregation of serumHBeAg. By day nine, the mice that received the specificity exchangerpeptide (SEQ. ID. No. 74) had a 3.3 fold increase in aggregation ofserum HBeAg levels. Similar to the 4-2 mAb, the specificity exchangerpeptide (SEQ. ID. No. 74) retained the ability to complex serum HBeAg invivo. These results verify that a specificity exchanger directed to anHBV antigen can be created and that such an agent can appreciablyaggregate HBcAg and/or HBeAg in vivo. TABLE 18 In vivo aggregation ofserum HBeAg in transgenic mice* Fold change in serum HBeAg levels mAbgiven as determined by EIA in HBeAg-Tg in vivo mice at indicated dayfrom injection Day 0 1 3 6 9 RPMI 1 1.2 1.7 1.4 1.7 HSV mAb 1 1.2 1.91.5 1.7 9C8 1 1.4 2.0 1.7 1.8 Peptide of 1 1.5 2.3 2.3 3.3 SEQ. ID. No.74

[0149] Another particularly desirable characterization assay evaluatesspecificity exchangers in chimpanzees infected with HBV. The nextexample describes this characterization assay in detail.

EXAMPLE 13

[0150] An approach to evaluate the efficacy of a specificity exchangerin chimpanzees is provided below. Accordingly, chimpanzees arerepeatedly inoculated with an antigen that is known to promote a hightiter antibody response (e.g., HSVgG2). After the course ofimmunization, the presence of polyclonal antibodies to the antigen isverified. Subsequently, the chimpanzee is infected with HBV and after astable infection is verified, the infected animal is provided atherapeutically effective dose of the specificity exchanger (SEQ. ID.No. 74). The dosages used in various animals include 100 μg/kg bodyweight to 500 μg/kg body weight. Aggregation of HBcAg and/or HBeAg isverified and the clearance of viral particles and long term monitoringof infection is conducted by analyzing blood samples for the presence ofHBV nucleic acid or protein by using the techniques described in Example10. Over a period of several treatments with the specificity exchanger,a reduction in viral lode will be observed.

[0151] Characterization assays also include experiments that evaluatethe ability of a binding partner to modulate a host immune response toHBV. It is contemplated that HBV reduces the CTL response of an infectedhost by targeting HBcAg to a high number of B cells, which then processthe antigen. Through antigen leakage, HBcAg-peptides are present on theclass I molecules of B cells whereby the CTL response against HBcAg andHBV is inhibited. To prevent this molecular cascade and, thereby,modulate a host immune response to HBV, binding partners that inhibitthe binding of HBcAg to B cells can be provided so as to preventtolerization of the HBcAg-specific CTL response of an infected host.Subjects infected with HBV have antigen presenting cells (e.g.,dendritic cells and B cells) that display HBV viral antigens and effectT cell proliferation. By analyzing the ability of binding partners tomodulate B cell presentation of HBV antigens in vitro, for example, onecan accurately determine whether a binding partner will modulate a hostimmune response to HBV. Accordingly, this type of characterization assayis performed by obtaining “naïve” B cells (i.e., B cells from animalsthat have not come in contact with HBV or an HBV antigen) and contactingthe naïve B cells with “experienced” T cells (i.e., T cells from animalsthat have been immunized with a HBV viral antigen or infected with HBV)in the presence of a binding agent. The ability of the binding agent tomodulate a host immune response to HBV is then determined by monitoringthe production of cytokines and/or T cell proliferation. The examplebelow describes an assay that was used to determine the ability of abinding partner to modulate an immune response in a subject.

EXAMPLE 14

[0152] A characterization assay that was used to evaluate the ability ofa binding partner to modulate a host immune system response to HBV wasconducted as follows. Groups of Balb/c mice were immunizedsubcutaneously with 20 μg HBcAg. Ten days later, draining lymph nodeswere harvested and experienced CD4⁺ T cells were purified using anti-CD4coated magnetic beads (Dynal AS, Oslo, Norway) according to themanufacturers instructions. Naïve B cells were obtained from syngeneicmice, which had not been immunized. These B cells were used as antigenpresenting cells in the characterization assay.

[0153] The ability of the different mAbs and peptides to block B celluptake and antigen presentation of HBcAg-peptides to HBcAg-specific CD4⁺T cells was then tested in vitro. The cell populations were mixed at aB/T cell ratio of 1:5 and added at a final concentration ofapproximately 2.5×10⁵ to 5.0×10⁵ cells per well in a 96 well microplate.Approximately, 20 μg of HBcAg was preincubated with the binding partner.The binding partner mixtures were then added to the cells and the plateswere incubated for 26 to 96 hours at 37° C. The effect of the bindingpartners on T cell proliferation was determined by monitoring [³H]thymidine incorporation.

[0154] As shown in TABLE 19, mAb 9C8 and mAb 4-2 and the HBcAg and HBeAgbinding peptide (SEQ.ID. No. 74) inhibited B cell mediated antigenpresentation of HBcAg to specific CD4⁺ T cells. In contrast, the controlpeptide (SEQ.ID. No. 19) showed no inhibition. Thus, the specificityexchanger (SEQ.ID. No. 74) not only bound HBcAg and HBeAg but alsopossessed the ability to block uptake and antigen presentation of HBcAgby B cells. TABLE 19 Inhibition of B cell - mediated antigenpresentation of HBcAg to experienced T cells* % Cell inhibition*population Antigen Inhibitor cpm SD of APC T cells 20 μg none 82 30Negative HBcAg control T + B cells none none 110 46 backgroundproliferation T + B cells 20 μg none 573 28 Positive HBcAg control T + Bcells 20 μg Mab 9C8 20 1 100 HBcAg T + B cells 20 μg MAb 4-2 245 123 71HBcAg T + B cells 20 μg 4-2 peptide 345 8 49 HBcAg (SEQ. ID. No. 74) T +B cells 20 μg Control peptide 572 183 0 HBcAg (SEQ. ID. No. 19)

[0155] It is contemplated that some binding partners will be highlyefficient B cell stimulatory molecules in that they effect a rapid andpotent T cell response. Other binding partners are contemplated toweakly activate antigen presenting cells and, thus, stimulate a weak Tcell response, if any at all. Classes of such weak and strong bindingpartners can be created based on similarities in structure and function.These classes and profiles can be entered onto a computer readablemedia, placed in a database, and accessed for comparison so as todevelop more effective weak and strong binding partners. The sectionbelow describes the use of binding partners as biotechnological toolsand diagnostic reagents.

[0156] Biotechnological Tools and Diagnostic Reagents

[0157] In one aspect , binding partners are used as biotechnologicaltools that detect the presence or absence, as well as the concentrationof HBcAg or HBeAg in a biological sample. The peptides can be used inmany different immunohistochemical techniques including but not limitedto, immunoprecipitation, Western blot, affinity purification, and insitu analysis. Advantageously, some embodiments can be used as highaffinity probes that detect HBV in tissues that are difficult to labelusing conventional antibodies.

[0158] Desirably, the binding partners are used as diagnostic reagentsto determine the presence of HBV infection in a subject or to monitorthe treatment of HBV infection in a subject. Further, the manufacture ofkits that incorporate the binding partners are contemplated. Thedetection component of these kits will typically be supplied incombination with one or more of the following reagents. A supportcapable of absorbing or otherwise binding protein will often besupplied. Available supports include membranes of nitrocellulose, nylonor derivatized nylon that can be characterized by bearing an array ofpositively charged substituents. One or more control reagents, buffers,enzymes, and detection material (e.g., radioisotope, enzyme conjugateand substrate, magnetic particle, gold particle, or secondary antibodywith or without conjugate) can be supplied in these kits.

[0159] The presence of HBV in a protein sample can be detected by usingconventional assays and a binding partner or specificity exchanger. Insome embodiments, a binding partner or specificity exchanger is used toimmunoprecipitate HBV viral antigens from solution or are used to reactwith HBV viral antigens on Western or Immunoblots. Favored diagnosticembodiments also include enzyme-linked immunosorbant assays (ELISA),radioimmunoassays (RIA), immunoradiometric assays (IRMA) andimmunoenzymatic assays (IEMA), including sandwich assays usingmonoclonal and/or polyclonal antibodies. Exemplary sandwich assays aredescribed by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530,hereby incorporated by reference. Other embodiments employ aspects ofthe immune-strip technology disclosed in U.S. Pat. Nos. 5,290,678;5,604,105; 5,710,008; 5,744,358; and 5,747,274, herein incorporated byreference.

[0160] In another preferred protein-based diagnostic, binding partnersor specificity exchangers are attached to a support in an ordered array,wherein a plurality of binding partners or specificity exchangers areattached to distinct regions of the support that do not overlap witheach other. These arrays are designed to be “addressable” such that thedistinct locations are recorded and can be accessed as part of an assayprocedure. The binding partners or specificity exchangers (collectivelyreferred to as “probes” in this context) are joined to the support indifferent known locations. The knowledge of the precise location of eachprobe makes these “addressable” arrays particularly useful in bindingassays. For example, an addressable array can comprise a support havingseveral regions to which are joined a plurality of probes thatspecifically recognize the presence of HBcAg and/or HBeAg in abiological sample obtained from subjects suspected of having contactwith HBV.

[0161] Accordingly, proteins are recovered from biological samples fromsubjects suspected of contracting HBV and are labeled by conventionalapproaches (e.g., radioactivity, calorimetrically, or fluorescently).The labeled protein samples are then applied to the array underconditions that permit binding to the probes. If a protein in the samplebinds to a probe on the array, then a signal will be detected at aposition on the support that corresponds to the location of theprobe-protein complex. Since the identity of each labeled sample isknown and the region of the support on which the labeled sample wasapplied is known, an identification of the presence of HBV infection canbe rapidly determined. These approaches are easily automated usingtechnology known to those of skill in the art of high throughputdiagnostic analysis.

[0162] In another embodiment, an opposite approach to that presentedabove can be employed. Proteins present in biological samples can bedisposed on a support so as to create an addressable array. Preferably,the protein samples are disposed on the support at known positions thatdo not overlap. The presence of a viral antigen in each sample is thendetermined by applying labeled probes that recognize HBcAg and/or HBeAg.Because the identity of the biological sample and its position on thearray is known, an identification of the presence of HBV infection canbe rapidly determined. As detailed above, any addressable arraytechnology known in the art can be employed with this aspect and displaythe protein arrays on the chips in an attempt to maximize antibodybinding patterns and diagnostic information.

[0163] Although many embodiments were chemically synthesized usingconventional techniques in peptide chemistry, nucleic acids encoding thepeptides can be introduced into cells in vitro or in vivo and therecipient cells can be made to express a binding partner, preferably aspecificity exchanger. A description of several approaches to make cellsthat express a binding partner is given in the section below.

[0164] Cells Made to Express Binding Partners

[0165] Cells made to express a binding partner, whether in vivo or invitro, are embodiments of the invention. The concentration of a bindingpartner, preferably a specificity exchanger, can be raised in a cell invitro by transfecting expression constructs encoding these molecules. Invivo expression constructs can also be used to deliver a nucleic acidencoding a binding partner to liver cells in an animal. Liposomemediated transfer can also be used to transfer a nucleic acid encoding abinding partner to a cell in vivo or in vitro.

[0166] The following is provided as one possible method to express abinding partner or specificity exchanger in a cell in vitro. First, themethionine initiation codon for a binding partner or specificityexchanger and the poly A signal of the gene are identified. If thenucleic acid encoding the polypeptide to be expressed lacks a methionineto serve as the initiation site, an initiating methionine can beintroduced next to the first codon of the nucleic acid usingconventional techniques. Similarly, if the nucleic acid lacks a poly Asignal, this sequence can be added to the construct by, for example,splicing out the Poly A signal from pSG5 (Stratagene) using BglI andSalI restriction endonuclease enzymes and incorporating it into themammalian expression vector pXT1 (Stratagene). The vector pXT1 containsthe LTRs and a portion of the gag gene from Moloney Murine LeukemiaVirus. The position of the LTRs in the construct allow efficient stabletransfection. The vector includes the Herpes Simplex Thymidine Kinasepromoter and the selectable neomycin gene.

[0167] The nucleic acid encoding the polypeptide to be expressed can beobtained by PCR from a bacterial vector having the binding partner usingoligonucleotide primers complementary to the nucleic acid and containingrestriction endonuclease sequences for Pst I incorporated into the5′primer and BglII at the 5′ end of the corresponding cDNA 3′ primer,taking care to ensure that the nucleic acid is positioned in frame withthe poly A signal. The purified fragment obtained from the resulting PCRreaction is digested with PstI, blunt ended with an exonuclease,digested with BglII, purified and ligated to pXT1, now containing a polyA signal and digested with BglII. The ligated product is transfectedinto a suitable cell line using Lipofectin (Life Technologies, Inc.,Grand Island, New York) under conditions outlined in the productspecification. Positive transfectants are selected after growing thetransfected cells in 600 ug/ml G418 (Sigma, St. Louis, Mo.). Preferablythe expressed protein is released into the culture medium, therebyfacilitating purification.

[0168] Another approach utilizes the “Xpress system for expression andpurification” (Invitrogen, San Diego, Calif.). The Xpress system isdesigned for high-level production and purification of recombinantproteins from bacterial, mammalian, and insect cells. The Xpress vectorsproduce recombinant proteins fused to a short N-terminal leader peptidethat has a high affinity for divalent cations. Using a nickel-chelatingresin (Invitrogen), the recombinant protein can be purified in one stepand the leader can be subsequently removed by cleavage withenterokinase.

[0169] One preferred vector for the expression of binding partners andfragments of binding partner is the pBlueBacHis2 Xpress. ThepBlueBacHis2 Xpress vector is a Baculovirus expression vector containinga multiple cloning site, an ampicillin resistance gene, and a Lac Zgene. By one approach, the binding partner or specificity exchangernucleic acid is cloned into the pBlueBacHis2 Xpress vector and SF9 cellsare infected. The expression protein is then isolated or purifiedaccording to the manufacturer's instructions. Several other culturedcell lines having recombinant constructs or vectors comprising a bindingpartner or specificity exchanger are embodiments and their manufacturewould be routine given the present disclosure.

[0170] By similar approaches, a nucleic acid encoding a binding partnercan be incorporated into a vector that expresses the binding partner orspecificity exchanger in liver cells in vivo. (Huber et al. Proc. Natl.Acad. Sci. USA 88:8039-8043(1991), herein expressly incorporated byreference in its entirety. Many such organ specific vectors have beendescribed in the literature and nucleic acids encoding a binding partneror specificity exchanger can be incorporated into these vectors byconventional techniques in molecular biology. (See U.S. Pat. Nos.5,981,274; 5,998,205; and 6,025,195, all of which are hereinincorporated by reference in their entirety.) In the disclosure below,several pharmaceutical embodiments are described.

[0171] Pharmaceutical Preparations and Methods of Administration

[0172] Binding partners, preferably a specificity exchanger, aresuitable for incorporation into pharmaceuticals for administration tosubjects in need of a compound that treats or prevents HBV infection.These pharmacologically active compounds can be processed in accordancewith conventional methods of galenic pharmacy to produce medicinalagents for administration to mammals including humans. The activeingredients can be incorporated into a pharmaceutical product with andwithout modification. Further, the manufacture of pharmaceuticals ortherapeutic agents that deliver the pharmacologically active compoundsof this invention by several routes are aspects . For example, and notby way of limitation, DNA, RNA, and viral vectors having sequenceencoding a binding partner or specificity exchanger are used withembodiments. Nucleic acids encoding a binding partner or specificityexchanger can be administered alone or in combination with other activeingredients.

[0173] The compounds can be employed in admixture with conventionalexcipients, i.e., pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral, enteral (e.g., oral) ortopical application that do not deleteriously react with thepharmacologically active ingredients described herein. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohols, gum arabic, vegetable oils, benzylalcohols, polyetylene glycols, gelatine, carbohydrates such as lactose,amylose or starch, magnesium stearate, talc, silicic acid, viscousparaffin, perfume oil, fatty acid monoglycerides and diglycerides,pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinylpyrrolidone, etc. Many more suitable vehicles are described inRemmington's Pharmaceutical Sciences, 15th Edition, Easton:MackPublishing Company, pages 1405-1412 and 1461-1487(1975) and The NationalFormulary XIV, 14th Edition, Washington, American PharmaceuticalAssociation (1975), herein incorporated by reference. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like that do notdeleteriously react with the active compounds.

[0174] The effective dose and method of administration of a particularpharmaceutical formulation having a binding partner or specificityexchanger can vary based on the individual needs of the patient and thetreatment or preventative measure sought. Therapeutic efficacy andtoxicity of such compounds can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose therapeutically effective in 50% of the population). For example, abinding partner or specificity exchanger can be evaluated using thecharacterization assays described above. The data obtained from theseassays is then used in formulating a range of dosage for use with otherorganisms, including humans. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED50 with no toxicity. The dosage varies within this range dependingupon type of binding partner or specificity exchanger, the dosage formemployed, sensitivity of the organism, and the route of administration.

[0175] Normal dosage amounts of a binding partner or specificityexchanger can vary from approximately 1 to 100,000 micrograms, up to atotal dose of about 10 grams, depending upon the route ofadministration. Desirable dosages include about 250 μg-1 mg, about 50mg-200mg, and about 250 mg-500 mg.

[0176] In some embodiments, the dose of a binding partner or specificityexchanger preferably produces a tissue or blood concentration or bothfrom approximately 0.1 μM to 500 mM. Desirable doses produce a tissue orblood concentration or both of about 1 to 800 μM. Preferable dosesproduce a tissue or blood concentration of greater than about 10 μM toabout 500 μM. Although doses that produce a tissue concentration ofgreater than 800 μM are not preferred, they can be used with someembodiments . A constant infusion of a binding partner or specificityexchanger can also be provided so as to maintain a stable concentrationin the tissues as measured by blood levels.

[0177] The exact dosage is chosen by the individual physician in view ofthe patient to be treated. Dosage and administration are adjusted toprovide sufficient levels of the active moiety or to maintain thedesired effect. Additional factors that can be taken into accountinclude the severity of the disease, age of the organism, and weight orsize of the organism; diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Short acting pharmaceutical compositions are administered dailyor more frequently whereas long acting pharmaceutical compositions areadministered every 2 or more days, once a week, or once every two weeksor even less frequently.

[0178] Routes of administration of the pharmaceuticals include, but arenot limited to, topical, transdermal, parenteral, gastrointestinal,transbronchial, and transalveolar. Transdermal administration isaccomplished by application of a cream, rinse, gel, etc. capable ofallowing the pharmacologically active compounds to penetrate the skin.Parenteral routes of administration include, but are not limited to,electrical or direct injection such as direct injection into a centralvenous line, intravenous, intramuscular, intraperitoneal, intradermal,or subcutaneous injection. Gastrointestinal routes of administrationinclude, but are not limited to, ingestion and rectal. Transbronchialand transalveolar routes of administration include, but are not limitedto, inhalation, either via the mouth or intranasally.

[0179] Compositions having pharmacologically active compounds describedherein that are suitable for transdermal or topical administrationinclude, but are not limited to, pharmaceutically acceptablesuspensions, oils, creams, and ointments applied directly to the skin orincorporated into a protective carrier such as a transdermal device(“transdermal patch”). Examples of suitable creams, ointments, etc. canbe found, for instance, in the Physician's Desk Reference. Examples ofsuitable transdermal devices are described, for instance, in U.S. Pat.No. 4,818,540 issued Apr. 4, 1989 to Chinen, et al., herein incorporatedby reference.

[0180] Compositions having pharmacologically active compounds that aresuitable for parenteral administration include, but are not limited to,pharmaceutically acceptable sterile isotonic solutions. Such solutionsinclude, but are not limited to, saline and phosphate buffered salinefor injection into a central venous line, intravenous, intramuscular,intraperitoneal, intradermal, or subcutaneous injection.

[0181] Compositions having pharmacologically active compounds that aresuitable for transbronchial and transalveolar administration include,but not limited to, various types of aerosols for inhalation. Devicessuitable for transbronchial and transalveolar administration of theseare also embodiments. Such devices include, but are not limited to,atomizers and vaporizers. Many forms of currently available atomizersand vaporizers can be readily adapted to deliver compositions having thepharmacologically active compounds.

[0182] Compositions having pharmacologically active compounds that aresuitable for gastrointestinal administration include, but not limitedto, pharmaceutically acceptable powders, pills or liquids for ingestionand suppositories for rectal administration. Due to the ease of use,gastrointestinal administration, particularly oral, is a preferredembodiment. Once the pharmaceutical comprising the binding partner orspecificity exchanger has been obtained, it can be administered to aorganism in need to treat or prevent HBV infection.

[0183] Aspects of the invention also include a coating for medicalequipment such as prosthetics, implants, and instruments. Coatingssuitable for use in medical devices can be provided by a gel or powdercontaining the binding partners or by polymeric coating into which abinding partner is suspended. Suitable polymeric materials for coatingsor devices are those that are physiologically acceptable and throughwhich a therapeutically effective amount of the binding partner candiffuse. Suitable polymers include, but are not limited to,polyurethane, polymethacrylate, polyamide, polyester, polyethylene,polypropylene, polystyrene, polytetrafluoroethylene, polyvinyl-chloride,cellulose acetate, silicone elastomers, collagen, silk, etc. Suchcoatings are described, for instance, in U.S. Pat. No. 4,612,337, issuedSep. 16, 1986 to Fox et al. that is incorporated herein by reference inits entirety.

[0184] In several aspects, a pharmaceutical having a binding partner,preferably a specificity exchanger, is provided to a subject in need ofan agent that treats or prevents HBV infection. These pharmaceuticalscan be formulated with or without a carrier or other agent in additionto the active ingredient, as described above. Methods to formulate suchpharmaceuticals that inhibit HBV infection and/or modulate a host immuneresponse to HBV are embodiments.

[0185] Other embodiments involve methods to treat or prevent HBVinfection. Accordingly, a subject in need of a binding partner orspecificity exchanger that inhibits HBV infection and/or modulates ahost immune response to HBV is provided a therapeutically effectiveamount of a pharmaceutical having said binding partner or specificityexchanger. Such subjects in need can include individuals at risk ofcontracting HBV or are already afflicted with HBV. These individuals canbe identified by clinical or biochemical techniques.

[0186] Although the invention has been described with reference toembodiments and examples, it should be understood that variousmodifications can be made without departing from the spirit.Accordingly, the invention is limited only by the following claims. Allreferences cited herein are hereby expressly incorporated by reference.

1 111 1 119 PRT Artificial Sequence Artificial Peptide 1 Val Lys Leu GlnGln Ser Gly Thr Glu Val Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys LeuSer Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr Asp 20 25 30 Ile Asp TrpVal Arg Gln Thr Pro Glu Gln Gly Leu Glu Trp Ile Gly 35 40 45 Trp Ile PhePro Gly Glu Gly Ser Thr Glu Tyr Asn Glu Lys Phe Lys 50 55 60 Gly Arg AlaThr Leu Ser Val Asp Lys Ser Ser Ser Thr Ala Tyr Met 65 70 75 80 Glu LeuThr Arg Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala 85 90 95 Arg GlyAsp Tyr Asp Tyr Tyr Arg Arg Tyr Phe Asp Leu Trp Gly Gln 100 105 110 GlyThr Thr Val Thr Val Ser 115 2 129 PRT Artificial Sequence ArtificialPeptide 2 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser LeuGly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val SerThr Ser 20 25 30 Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly GlnPro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly ValPro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu AsnIle His 65 70 75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys GlnHis Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu GluIle Lys Arg 100 105 110 Ala Asp Ala Ala Pro Ala Val Ser Ile Phe Pro ProSer Ser Lys Leu 115 120 125 Gly 3 130 PRT Artificial Sequence ArtificialPeptide 3 Ile Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly AlaSer 1 5 10 15 Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr GlyTyr Asn 20 25 30 Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu TrpIle Gly 35 40 45 Asn Ile Asn Pro Tyr Tyr Gly Ser Thr Ser Tyr Asn Gln LysPhe Lys 50 55 60 Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr AlaTyr Met 65 70 75 80 Gln Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Val TyrTyr Cys Ala 85 90 95 Arg Gly Lys Gly Thr Gly Phe Ala Tyr Trp Gly Gln GlyThr Leu Val 100 105 110 Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser ValTyr Pro Leu Val 115 120 125 Pro Val 130 4 20 PRT Artificial SequenceArtificial Peptide 4 Val Lys Leu Gln Gln Ser Gly Thr Glu Val Val Lys ProGly Ala Ser 1 5 10 15 Val Lys Leu Ser 20 5 20 PRT Artificial SequenceArtificial Peptide 5 Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys AlaSer Gly Tyr 1 5 10 15 Ile Phe Thr Ser 20 6 20 PRT Artificial SequenceArtificial Peptide 6 Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr Asp IleAsp Trp Val 1 5 10 15 Arg Gln Thr Pro 20 7 20 PRT Artificial SequenceArtificial Peptide 7 Tyr Asp Ile Asp Trp Val Arg Gln Thr Pro Glu Gln GlyLeu Glu Trp 1 5 10 15 Ile Gly Trp Ile 20 8 20 PRT Artificial SequenceArtificial Peptide 8 Glu Gln Gly Leu Glu Trp Ile Gly Trp Ile Phe Pro GlyGlu Gly Ser 1 5 10 15 Thr Glu Tyr Asn 20 9 20 PRT Artificial SequenceArtificial Peptide 9 Phe Pro Gly Glu Gly Ser Thr Glu Tyr Asn Glu Lys PheLys Gly Arg 1 5 10 15 Ala Thr Leu Ser 20 10 20 PRT Artificial SequenceArtificial Peptide 10 Glu Lys Phe Lys Gly Arg Ala Thr Leu Ser Val AspLys Ser Ser Ser 1 5 10 15 Thr Ala Tyr Met 20 11 20 PRT ArtificialSequence Artificial Peptide 11 Val Asp Lys Ser Ser Ser Thr Ala Tyr MetGlu Leu Thr Arg Leu Thr 1 5 10 15 Ser Glu Asp Ser 20 12 20 PRTArtificial Sequence Artificial Peptide 12 Glu Leu Thr Arg Leu Thr SerGlu Asp Ser Ala Val Tyr Phe Cys Ala 1 5 10 15 Arg Gly Asp Tyr 20 13 20PRT Artificial Sequence Artificial Peptide 13 Ala Val Tyr Phe Cys AlaArg Gly Asp Tyr Asp Tyr Tyr Arg Arg Tyr 1 5 10 15 Phe Asp Leu Trp 20 1419 PRT Artificial Sequence Artificial Peptide 14 Asp Tyr Tyr Arg Arg TyrPhe Asp Leu Trp Gly Gln Gly Thr Thr Val 1 5 10 15 Thr Val Ser 15 20 PRTArtificial Sequence Artificial Peptide 15 Asp Ile Val Leu Thr Gln SerPro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr 20 16 20PRT Artificial Sequence Artificial Peptide 16 Leu Ala Val Ser Leu GlyGln Arg Ala Thr Ile Ser Cys Arg Ala Ser 1 5 10 15 Gln Ser Val Ser 20 1720 PRT Artificial Sequence Artificial Peptide 17 Ile Ser Cys Arg Ala SerGln Ser Val Ser Thr Ser Ser Tyr Ser Tyr 1 5 10 15 Met His Trp Tyr 20 1820 PRT Artificial Sequence Artificial Peptide 18 Thr Ser Ser Tyr Ser TyrMet His Trp Tyr Gln Gln Lys Pro Gly Gln 1 5 10 15 Pro Pro Lys Leu 20 1920 PRT Artificial Sequence Artificial Peptide 19 Gln Gln Lys Pro Gly GlnPro Pro Lys Leu Leu Ile Lys Tyr Ala Ser 1 5 10 15 Asn Leu Glu Ser 20 2020 PRT Artificial Sequence Artificial Peptide 20 Leu Ile Lys Tyr Ala SerAsn Leu Glu Ser Gly Val Pro Ala Arg Phe 1 5 10 15 Ser Gly Ser Gly 20 2120 PRT Artificial Sequence Artificial Peptide 21 Gly Val Pro Ala Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr 1 5 10 15 Leu Asn Ile His 20 2220 PRT Artificial Sequence Artificial Peptide 22 Ser Gly Thr Asp Phe ThrLeu Asn Ile His Pro Val Glu Glu Glu Asp 1 5 10 15 Thr Ala Thr Tyr 20 2320 PRT Artificial Sequence Artificial Peptide 23 Pro Val Glu Glu Glu AspThr Ala Thr Tyr Tyr Cys Gln His Ser Trp 1 5 10 15 Glu Ile Pro Tyr 20 2420 PRT Artificial Sequence Artificial Peptide 24 Tyr Cys Gln His Ser TrpGlu Ile Pro Tyr Thr Phe Gly Gly Gly Thr 1 5 10 15 Lys Leu Glu Ile 20 2519 PRT Artificial Sequence Artificial Peptide 25 Thr Phe Gly Gly Gly ThrLys Leu Glu Ile Lys Arg Ala Asp Ala Ala 1 5 10 15 Pro Ala Val 26 19 PRTArtificial Sequence Artificial Peptide 26 Lys Arg Ala Asp Ala Ala ProAla Val Ser Ile Phe Pro Pro Ser Ser 1 5 10 15 Lys Leu Gly 27 20 PRTArtificial Sequence Artificial Peptide 27 Ile Gln Leu Gln Gln Ser GlyAla Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser 20 28 20PRT Artificial Sequence Artificial Peptide 28 Val Lys Pro Gly Ala SerVal Lys Ile Ser Cys Lys Ala Ser Gly Tyr 1 5 10 15 Ser Phe Thr Gly 20 2920 PRT Artificial Sequence Artificial Peptide 29 Cys Lys Ala Ser Gly TyrSer Phe Thr Gly Tyr Asn Met Asn Trp Val 1 5 10 15 Lys Gln Ser His 20 3020 PRT Artificial Sequence Artificial Peptide 30 Tyr Asn Met Asn Trp ValLys Gln Ser His Gly Lys Ser Leu Glu Trp 1 5 10 15 Ile Gly Asn Ile 20 3120 PRT Artificial Sequence Artificial Peptide 31 Gly Lys Ser Leu Glu TrpIle Gly Asn Ile Asn Pro Tyr Tyr Gly Ser 1 5 10 15 Thr Ser Tyr Asn 20 3220 PRT Artificial Sequence Artificial Peptide 32 Asn Pro Tyr Tyr Gly SerThr Ser Tyr Asn Gln Lys Phe Lys Gly Lys 1 5 10 15 Ala Thr Leu Thr 20 3320 PRT Artificial Sequence Artificial Peptide 33 Gln Lys Phe Lys Gly LysAla Thr Leu Thr Val Asp Lys Ser Ser Ser 1 5 10 15 Thr Ala Tyr Met 20 3420 PRT Artificial Sequence Artificial Peptide 34 Val Asp Lys Ser Ser SerThr Ala Tyr Met Gln Leu Asn Ser Leu Thr 1 5 10 15 Ser Glu Asp Ser 20 3520 PRT Artificial Sequence Artificial Peptide 35 Gln Leu Asn Ser Leu ThrSer Glu Asp Ser Ala Val Tyr Tyr Cys Ala 1 5 10 15 Arg Gly Lys Gly 20 3620 PRT Artificial Sequence Artificial Peptide 36 Ala Val Tyr Tyr Cys AlaArg Gly Lys Gly Thr Gly Phe Ala Tyr Trp 1 5 10 15 Gly Gln Gly Thr 20 3720 PRT Artificial Sequence Artificial Peptide 37 Thr Gly Phe Ala Tyr TrpGly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5 10 15 Ala Lys Thr Thr 20 3820 PRT Artificial Sequence Artificial Peptide 38 Leu Val Thr Val Ser AlaAla Lys Thr Thr Pro Pro Ser Val Tyr Pro 1 5 10 15 Leu Val Pro Val 20 3919 PRT Artificial Sequence Artificial Peptide 39 Lys Pro Gly Ala Ser ValLys Leu Ser Cys Lys Ala Ser Gly Tyr Ile 1 5 10 15 Phe Thr Ser 40 18 PRTArtificial Sequence Artificial Peptide 40 Pro Gly Ala Ser Val Lys LeuSer Cys Lys Ala Ser Gly Tyr Ile Phe 1 5 10 15 Thr Ser 41 17 PRTArtificial Sequence Artificial Peptide 41 Gly Ala Ser Val Lys Leu SerCys Lys Ala Ser Gly Tyr Ile Phe Thr 1 5 10 15 Ser 42 16 PRT ArtificialSequence Artificial Peptide 42 Ala Ser Val Lys Leu Ser Cys Lys Ala SerGly Tyr Ile Phe Thr Ser 1 5 10 15 43 15 PRT Artificial SequenceArtificial Peptide 43 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr IlePhe Thr Ser 1 5 10 15 44 14 PRT Artificial Sequence Artificial Peptide44 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 10 45 13PRT Artificial Sequence Artificial Peptide 45 Lys Leu Ser Cys Lys AlaSer Gly Tyr Ile Phe Thr Ser 1 5 10 46 12 PRT Artificial SequenceArtificial Peptide 46 Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 15 10 47 11 PRT Artificial Sequence Artificial Peptide 47 Ser Cys Lys AlaSer Gly Tyr Ile Phe Thr Ser 1 5 10 48 10 PRT Artificial SequenceArtificial Peptide 48 Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser 1 5 10 499 PRT Artificial Sequence Artificial Peptide 49 Lys Ala Ser Gly Tyr IlePhe Thr Ser 1 5 50 8 PRT Artificial Sequence Artificial Peptide 50 AlaSer Gly Tyr Ile Phe Thr Ser 1 5 51 7 PRT Artificial Sequence ArtificialPeptide 51 Ser Gly Tyr Ile Phe Thr Ser 1 5 52 6 PRT Artificial SequenceArtificial Peptide 52 Gly Tyr Ile Phe Thr Ser 1 5 53 19 PRT ArtificialSequence Artificial Peptide 53 Ser Cys Arg Ala Ser Gln Ser Val Ser ThrSer Ser Tyr Ser Tyr Met 1 5 10 15 His Trp Tyr 54 18 PRT ArtificialSequence Artificial Peptide 54 Cys Arg Ala Ser Gln Ser Val Ser Thr SerSer Tyr Ser Tyr Met His 1 5 10 15 Trp Tyr 55 17 PRT Artificial SequenceArtificial Peptide 55 Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr SerTyr Met His Trp 1 5 10 15 Tyr 56 16 PRT Artificial Sequence ArtificialPeptide 56 Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr Met His TrpTyr 1 5 10 15 57 15 PRT Artificial Sequence Artificial Peptide 57 SerGln Ser Val Ser Thr Ser Ser Tyr Ser Tyr Met His Trp Tyr 1 5 10 15 58 14PRT Artificial Sequence Artificial Peptide 58 Gln Ser Val Ser Thr SerSer Tyr Ser Tyr Met His Trp Tyr 1 5 10 59 13 PRT Artificial SequenceArtificial Peptide 59 Ser Val Ser Thr Ser Ser Tyr Ser Tyr Met His TrpTyr 1 5 10 60 12 PRT Artificial Sequence Artificial Peptide 60 Val SerThr Ser Ser Tyr Ser Tyr Met His Trp Tyr 1 5 10 61 11 PRT ArtificialSequence Artificial Peptide 61 Ser Thr Ser Ser Tyr Ser Tyr Met His TrpTyr 1 5 10 62 10 PRT Artificial Sequence Artificial Peptide 62 Thr SerSer Tyr Ser Tyr Met His Trp Tyr 1 5 10 63 9 PRT Artificial SequenceArtificial Peptide 63 Ser Ser Tyr Ser Tyr Met His Trp Tyr 1 5 64 7 PRTArtificial Sequence Artificial Peptide 64 Tyr Ser Tyr Met His Trp Tyr 15 65 6 PRT Artificial Sequence Artificial Peptide 65 Ser Tyr Met His TrpTyr 1 5 66 16 PRT Artificial Sequence Artificial Peptide 66 Pro Gly AlaSer Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe 1 5 10 15 67 18 PRTArtificial Sequence Artificial Peptide 67 Lys Pro Gly Asp Ser Leu ArgLeu Ser Cys Lys Ala Ser Gly Tyr Thr 1 5 10 15 Phe Ser 68 20 PRTArtificial Sequence Artificial Peptide 68 Val Lys Pro Gly Gly Ser LeuArg Leu Ser Cys Val Ala Ser Gly Phe 1 5 10 15 Thr Phe Ser Ser 20 69 19PRT Artificial Sequence Artificial Peptide 69 Lys Pro Gly Asp Ser LeuArg Leu Ser Cys Lys Gly Ser Gly Phe Thr 1 5 10 15 Phe Ser Asn 70 21 PRTArtificial Sequence Artificial Peptide 70 Lys Leu Ser Cys Lys Ala SerGly Tyr Ile Phe Thr Ser Glu His Arg 1 5 10 15 Gly Gly Pro Glu Glu 20 7121 PRT Artificial Sequence Artificial Peptide 71 Lys Leu Ser Cys Lys AlaSer Gly Tyr Ile Phe Thr Ser His Arg Gly 1 5 10 15 Gly Pro Glu Glu Phe 2072 21 PRT Artificial Sequence Artificial Peptide 72 Lys Leu Ser Cys LysAla Ser Gly Tyr Ile Phe Thr Ser Arg Gly Gly 1 5 10 15 Pro Glu Glu PheGlu 20 73 21 PRT Artificial Sequence Artificial Peptide 73 Val Lys LeuSer Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Glu His 1 5 10 15 Arg GlyGly Pro Glu 20 74 21 PRT Artificial Sequence Artificial Peptide 74 ValLys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser His Arg 1 5 10 15Gly Gly Pro Glu Glu 20 75 21 PRT Artificial Sequence Artificial Peptide75 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser His Arg 1 510 15 Gly Gly Pro Glu Glu 20 76 21 PRT Artificial Sequence ArtificialPeptide 76 Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser GlyGly 1 5 10 15 Pro Glu Glu Phe Glu 20 77 4 PRT Artificial SequenceArtificial Peptide 77 Cys Lys Ala Ser 1 78 4 PRT Artificial SequenceArtificial Peptide 78 Cys Arg Ala Ser 1 79 17 PRT Artificial SequenceArtificial Peptide 79 Gly Leu Tyr Ser Ser Ile Trp Leu Ser Pro Gly ArgSer Tyr Phe Glu 1 5 10 15 Thr 80 17 PRT Artificial Sequence ArtificialPeptide 80 Tyr Thr Asp Ile Lys Tyr Asn Pro Phe Thr Asp Arg Gly Glu GlyAsn 1 5 10 15 Met 81 17 PRT Artificial Sequence Artificial Peptide 81Asp Gln Asn Ile His Met Asn Ala Arg Leu Leu Ile Arg Ser Pro Phe 1 5 1015 Thr 82 17 PRT Artificial Sequence Artificial Peptide 82 Leu Ile ArgSer Pro Phe Thr Asp Pro Gln Leu Leu Val His Thr Asp 1 5 10 15 Pro 83 17PRT Artificial Sequence Artificial Peptide 83 Gln Lys Glu Ser Leu LeuPhe Pro Pro Val Lys Leu Leu Arg Arg Val 1 5 10 15 Pro 84 11 PRTArtificial Sequence Artificial Peptide 84 Pro Ala Leu Thr Ala Val GluThr Gly Ala Thr 1 5 10 85 8 PRT Artificial Sequence Artificial Peptide85 Ser Thr Leu Val Pro Glu Thr Thr 1 5 86 13 PRT Artificial SequenceArtificial Peptide 86 Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro IleLeu 1 5 10 87 9 PRT Artificial Sequence Artificial Peptide 87 Glu IlePro Ala Leu Thr Ala Val Glu 1 5 88 10 PRT Artificial Sequence ArtificialPeptide 88 Leu Glu Asp Pro Ala Ser Arg Asp Leu Val 1 5 10 89 8 PRTArtificial Sequence Artificial Peptide 89 His Arg Gly Gly Pro Glu GluPhe 1 5 90 7 PRT Artificial Sequence Artificial Peptide 90 His Arg GlyGly Pro Glu Glu 1 5 91 17 PRT Artificial Sequence Artificial Peptide 91Val Leu Ile Cys Gly Glu Asn Thr Val Ser Arg Asn Tyr Ala Thr His 1 5 1015 Ser 92 17 PRT Artificial Sequence Artificial Peptide 92 Lys Ile AsnThr Met Pro Pro Phe Leu Asp Thr Glu Leu Thr Ala Pro 1 5 10 15 Ser 93 17PRT Artificial Sequence Artificial Peptide 93 Pro Asp Glu Lys Ser GlnArg Glu Ile Leu Leu Asn Lys Ile Ala Ser 1 5 10 15 Tyr 94 17 PRTArtificial Sequence Artificial Peptide 94 Thr Ala Thr Thr Thr Thr TyrAla Tyr Pro Gly Thr Asn Arg Pro Pro 1 5 10 15 Val 95 8 PRT ArtificialSequence Artificial Peptide 95 Ser Thr Pro Leu Pro Glu Thr Thr 1 5 96 20PRT Artificial Sequence Artificial Peptide 96 Ala Thr Val Glu Leu LeuSer Phe Leu Pro Ser Asp Phe Phe Pro Ser 1 5 10 15 Val Arg Asp Leu 20 9720 PRT Artificial Sequence Artificial Peptide 97 Ser Asp Phe Phe Pro SerVal Arg Asp Leu Leu Asp Thr Ala Ser Ala 1 5 10 15 Leu Tyr Arg Glu 20 9820 PRT Artificial Sequence Artificial Peptide 98 Leu Asp Thr Ala Ser AlaLeu Tyr Arg Glu Ala Leu Glu Ser Pro Glu 1 5 10 15 His Cys Ser Pro 20 9920 PRT Artificial Sequence Artificial Peptide 99 Ala Leu Glu Ser Pro GluHis Cys Ser Pro His His Thr Ala Leu Arg 1 5 10 15 Gln Ala Ile Leu 20 10020 PRT Artificial Sequence Artificial Peptide 100 His His Thr Ala LeuArg Gln Ala Ile Leu Cys Trp Gly Glu Leu Met 1 5 10 15 Thr Leu Ala Thr 20101 20 PRT Artificial Sequence Artificial Peptide 101 Cys Trp Gly GluLeu Met Thr Leu Ala Thr Trp Val Gly Val Asn Leu 1 5 10 15 Glu Asp ProAla 20 102 20 PRT Artificial Sequence Artificial Peptide 102 Trp Val GlyVal Asn Leu Glu Asp Pro Ala Ser Arg Asp Leu Val Val 1 5 10 15 Ser TyrVal Asn 20 103 20 PRT Artificial Sequence Artificial Peptide 103 Ser ArgAsp Leu Val Val Ser Tyr Val Asn Thr Asn Met Gly Leu Lys 1 5 10 15 PheArg Gln Leu 20 104 20 PRT Artificial Sequence Artificial Peptide 104 ThrAsn Met Gly Leu Lys Phe Arg Gln Leu Leu Trp Phe His Ile Ser 1 5 10 15Cys Leu Thr Phe 20 105 20 PRT Artificial Sequence Artificial Peptide 105Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Ile 1 5 1015 Glu Tyr Leu Val 20 106 20 PRT Artificial Sequence Artificial Peptide106 Gly Arg Glu Thr Val Ile Glu Tyr Leu Val Ser Phe Gly Val Trp Ile 1 510 15 Arg Thr Pro Pro 20 107 20 PRT Artificial Sequence ArtificialPeptide 107 Ser Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro ProAsn 1 5 10 15 Ala Pro Ile Leu 20 108 20 PRT Artificial SequenceArtificial Peptide 108 Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser ThrLeu Pro Glu Thr 1 5 10 15 Thr Val Val Arg 20 109 20 PRT ArtificialSequence Artificial Peptide 109 Ser Thr Leu Pro Glu Thr Thr Val Val ArgArg Arg Gly Arg Ser Pro 1 5 10 15 Arg Arg Arg Thr 20 110 20 PRTArtificial Sequence Artificial Peptide 110 Arg Arg Gly Arg Ser Pro ArgArg Arg Thr Pro Ser Pro Arg Arg Arg 1 5 10 15 Arg Ser Gln Ser 20 111 23PRT Artificial Sequence Artificial Peptide 111 Pro Ser Pro Arg Arg ArgArg Ser Gln Ser Pro Arg Arg Arg Arg Ser 1 5 10 15 Gln Ser Arg Glu SerGln Cys 20

What is claimed is:
 1. An isolated or purified peptide comprising theformula: X¹ _(n)CZASX² _(n), wherein: “X¹” is any amino acid “C” iscysteine; “Z” is lysine or arginine; “A” is alanine; “S” is serine; “X²”is any amino acid; and “n” is an integer and, wherein said peptide isless than 50 amino acids in length and specifically binds HBcAg and/orHBeAg.
 2. The peptide of claim 1, wherein the sequence is selected fromthe group consisting of SEQ. ID. Nos. 1-78.
 3. The peptide of claim 1,wherein “X¹ _(n)” or “X² _(n)” encodes an epitope of a pathogen or atoxin.
 4. The peptide of claim 1, wherein “X_(n)” or “X² _(n)” encodesan epitope on a herpes simplex virus (HSV).
 5. The peptide of claim 4,wherein the epitope comprises a fragment of a peptide having thesequence of SEQ. ID. Nos. 70-76.
 6. A nucleic acid encoding a peptidehaving a sequence selected from the group consisting of SEQ. ID. No.1-78.
 7. A method of making a pharmaceutical comprising: identifying abinding partner that interacts with HBcAg or HBeAg having a sequenceselected from the group consisting of SEQ. ID. Nos. 1-78; andincorporating a therapeutically effective amount of said binding partnerinto a pharmaceutical.
 8. A method of treatment or prevention of HBVinfection comprising: identifying a subject in need of a molecule thatinhibits HBV infection; and providing said subject with a bindingpartner that interacts with HBcAg or HBeAg having a sequence selectedfrom the group consisting of SEQ. ID. Nos. 1-78.
 9. A method ofidentifying a binding partner having a sequence that interacts withHBcAg or HBeAg comprising: providing a support comprising HBcAg orHBeAg; contacting the support with a candidate binding partner having asequence selected from the group consisting of SEQ. ID. Nos. 1-78; anddetecting a biological complex comprising HBcAg or HBeAg and saidcandidate binding partner, wherein detection of such complex indicatesthat said candidate binding partner is a binding partner interacts withHBcAg or HBeAg.
 10. A method of identifying a binding partner thatinhibits HBV infection comprising: providing a cell that is infectedwith HBV; contacting said cell with a candidate binding partner selectedfrom the group consisting of SEQ. ID. Nos. 1-78; and determining whetherthe presence of said candidate binding partner having a sequence isassociated with a decrease in HBV infection.
 11. A method of identifyinga binding partner that modulates an immune system response comprising:providing a naïve antigen presenting cell; contacting said naïve antigenpresenting cell with a candidate binding partner and a T cell thatreacts to HBcAg or HBeAg; and detecting an inhibition or enhancement ofT cell stimulation whereby said binding partner is identified.
 12. Themethod of claim 11, wherein the detection step is performed byevaluating a change in cytokine production or T cell proliferation. 13.The method of claim 11, wherein the candidate binding partner has asequence selected from the group consisting of SEQ. ID. Nos. 1-78.
 14. Amethod of determining the presence of HBV in a biological samplecomprising: providing a biological sample; providing a binding partnerthat binds to HBcAg and/or HBeAg, wherein said binding partner has asequence selected from the group consisting of SEQ. ID. Nos. 1-78; anddetermining whether said binding partner binds to HBcAg and/or HBeAg.15. A diagnostic kit for the detection of HBV infection comprising abinding partner, wherein said binding partner has a sequence selectedfrom the group consisting of SEQ. ID. Nos. 1-78.
 16. A method ofinhibiting B cell mediated processing and uptake of HBcAg and/or HBeAgcomprising: providing a binding partner selected from the groupconsisting of SEQ. ID. Nos. 1-78; and determining whether said bindingpartner inhibits B cell mediated processing and uptake of HBcAg and/orHBeAg.
 17. The method of claim 16, wherein the determination of whethersaid binding partner inhibits B cell mediated processing and uptake ofHBcAg and/or HBeAg is accomplished by performing an assay of T cellproliferation or cytokine production.
 18. An isolated or purifiedpeptide consisting essentially of the peptide of SEQ. ID. No. 103 or104.
 19. A nucleic acid consisting essentially of a nucleic acidsequence encoding the peptide sequence of SEQ. ID. No. 103 or
 104. 20.An isolated or purified biological complex comprising a peptidecomprising the sequence of SEQ. ID. No. 74 and a member selected fromthe group consisting of a peptide consisting essentially of the sequenceof SEQ. ID. Nos. 103 or 104, HBcAg, HBeAg, HBV capsid protein, and HBV.